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Endometriosis and atherosclerotic cardiovascular disease (ASCVD) are both essentially diseases of inflammation. It is well established that inflammation is the leading mechanism in the initiation and maintenance of vascular injury and in the development and progression of atherosclerosis. Thus, if women with endometriosis do indeed have increased general inflammation, they are at increased risk of developing microvascular dysfunction and atherosclerosis. Currently available evidence suggests that young female patients with proven endometriosis may be at a higher lifetime risk of developing cardiovascular disease; this may be unrecognized due to the relatively young age of women found to have endometriosis. Other mechanisms proposed to explain the link between endometriosis and ASCVD include similarities in the genetic underpinnings of each condition, including microRNA dysfunction and the association between endometriosis and early menopause, a risk for developing ASCVD. Although physicians today primarily focus on traditional risk factors when evaluating an individual female patient's risk of developing ASCVD, we believe that a history of endometriosis should be included as a possible risk factor and needs further exploration. A better understanding of the mechanisms linking endometriosis with ASCVD will hopefully guide the implementation of new therapies to mitigate the increased cardiovascular disease burden that patients with endometriosis might face., (Copyright © 2019 AAGL. Published by Elsevier Inc. All rights reserved.)

Previous studies have shown conflicting data regarding cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes, and considering the widespread overexpression of cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown that cyclin D1 and Cdk2 form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with cyclin D1 and Cdk2 failing to complex in its absence. These p21/cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of cyclin D1/Cdk2 complexes, and that cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer, are shown to be phosphorylated by cyclin D1/Cdk2 complexes. Additionally, polypyrimidine tract binding protein-associated splicing factor (PSF) is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either cyclin E or cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity.

The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.

Dequalinium (DEQ), a drug with both antimicrobial and anticancer activity, induced the formation of petite (respiration-deficient) mutants in the yeast Saccharomyces cerevisiae. DEQ was found to be approximately 50-fold more potent than ethidium bromide (EB) at inducing petites. Analysis of the DEQ-induced petite mutants indicated a complete loss of mitochondrial DNA (<1 copy/cell). Prior to the loss of mtDNA, DEQ caused cleavage of the mtDNA into a population of fragments 30-40kbp in size suggesting that this drug causes petites by inducing a breakdown of mtDNA. The selective effect of DEQ on yeast mtDNA may underlie the antifungal activity of this chemotherapeutic agent.

We have previously reported the presence of a DNA gyrase-like topoisomerase activity associated with the 35kb apicoplast DNA in the malarial parasite Plasmodium falciparum [Weissig V, Vetro-Widenhouse TS, Rowe TC. Topoisomerase II inhibitors induce cleavage of nuclear and 35kb plastid DNAs in the malarial parasite Plasmodium falciparum. DNA Cell Biol 1997;16:1483]. Sequences encoding polypeptides homologous to both the A and B subunits of bacterial DNA gyrase have been identified in the genome sequence of P. falciparum among data produced by the Malaria Genome Consortium and the University of Florida Malaria Gene Sequence Tag Project. Based on these findings, we have cloned and expressed a region of the Plasmodium vivax GyrB gene encoding a 43kDa polypeptide homologous to the ATP-binding domain of Escherichia coli DNA gyrase. The 43kDa PvGyrB polypeptide was found to have intrinsic ATPase activity with a K(m) of 0.27mM and a k(cat) of 0.051s(-1). The PvGyrB ATPase was also sensitive to the bacterial DNA gyrase inhibitor coumermycin. The implications of these findings are discussed.

A phase I/II clinical study evaluated 17 patients with refractory/recurrent acute leukemia treated with 1.5 mg/m2/day topotecan on days 1-3 followed by etoposide (100 mg/m2/day)+mitoxantrone (10 mg/m2/day) on days 4, 5 and 9, 10. Timed sequential chemotherapy using the topoisomerase I-inhibitor topotecan before the topoisomerase II-inhibitors, etoposide+mitoxantrone (T-EM) treatment is proposed to induce topoisomerase II protein levels and potentiate the cytotoxic activity of the topoisomerase II-directed drugs. Fourteen patients had refractory and three had recurrent acute leukemia. The majority of patients were heavily pre-treated with greater than three re-induction chemotherapy regimens. Ten patients responded to T-EM treatment (59%). Four of seventeen (24%) had a complete remission and one had a partial remission. Four additional patients (24%) who scored complete leukemia clearance had no evidence of disease with complete white and red blood cell recovery but with platelet counts less than 100,000. The lack of platelet recovery in one patient having a partial response was scored as a partial leukemia clearance. The toxicity profile included major non-hematological toxicity including grade 3 mucositis (29%) and neutropenic fever (65%). Paired measurements of intracellular levels of topoisomerase II isoforms alpha and beta in leukemia blast cells (bone marrow) collected before (day 0) and after topotecan treatment (day 4) showed that a relative increase of topoisomerase IIalpha (Topo IIalpha) > or = 40% strongly correlated with response after T-EM treatment. Increased Topo IIalpha levels also corresponded to increased DNA fragmentation. Two patients who had an increase of Topo IIalpha of 20-25% had either a PR or PLC while patients with a < 10% increase showed no response to T-EM treatment. We conclude that timed sequential chemotherapy using topotecan followed by etoposide+mitoxantrone is an effective regimen for patients with refractory acute leukemia, and demonstrate Topo IIalpha protein level increases after topotecan treatment.

Topoisomerase II (topo II) is an essential enzyme that alters DNA topology. This activity is important for a variety of chromosome functions including DNA replication, transcription, recombination, and chromosome condensation and segregation. Previously we localized topo II in mouse gametes and preimplantation embryos using isoform-specific antibodies demonstrating the presence of the enzyme in oocytes and embryos, but not sperm. To probe functions of topo II during preimplantation development, we treated mouse zygotes with 100 nM teniposide, and assessed embryo morphology and DNA replication. Teniposide blocked cleavage in 69% embryos; the remainder cleaved once but had abnormal nuclei. Teniposide-treated embryos were devoid of topo II immunofluorescence. Teniposide also prevented DNA replication, implicating topo II in this process. Embryos treated with a 2 hr pulse of teniposide recovered and developed to the blastocyst stage, indicating 100 nM teniposide did not induce apoptosis. To more specifically analyze topo IIalpha function, we treated zygotes with topo IIalpha-targeted antisense oligodeoxynucleotides. Most zygotes arrested at the 2-cell stage while controls developed into blastocysts indicating topo IIalpha is essential for preimplantation development. The absence of topo IIalpha, but not beta immunofluorescence in antisense-treated embryos confirms the specificity and impact of the treatment. In addition, topo IIalpha is newly synthesized at the 2-cell stage. These results establish an essential function for topo II in mouse preimplantation embryonic development., (Copyright 2002 Wiley-Liss, Inc.)

DNA topoisomerase II (topo II) is an essential enzyme that mediates a variety of chromosome activities including DNA replication, transcription, recombination, and chromosome condensation and segregation. Isoform-specific anti-topo II antibodies were used to determine the distribution of topo II alpha and beta in mouse gametes and embryos. Immunoblot analysis with two anti-topo IIalpha antibodies revealed that a 170 kDa topo IIalpha band was present in ovary and testis. Mature sperm exhibited an 89 kDa band only, which may be a degradation product of topo IIalpha. Immunoblots probed with a monoclonal antibody that recognizes both isoforms, showed bands at 170 and 180 kDa, which correspond to topo IIalpha and beta, respectively. An additional 100 kDa band was also present in ovary and testis. Mature sperm did not exhibit staining with this antibody. We also localized topo II in mouse gametes and embryos up to the blastocyst stage using immunofluorescence microscopy. While both isoforms were found in nuclei and nucleoli of germinal vesicle oocytes, topo IIalpha localized to metaphase chromosomes during meiosis, and only to nucleoli during embryonic interphase. Topo IIbeta was absent from chromosomes of metaphase II oocytes, but localized to embryonic interphase nuclei. Both full-length isoforms were absent from sperm, indicating topo II is stored maternally. These results identify topo II as an important component of mouse oocyte and embryonic chromatin, and suggest its involvement in oocyte maturation and preimplantation embryonic development. The different immunofluorescent staining patterns indicate topo IIalpha and beta may serve different roles during the embryonic cell cycle., (Copyright 2002 Wiley-Liss, Inc.)

Numerous drugs are known to deplete mitochondrial DNA (mtDNA) from mammalian cells. These include DNA polymerase gamma and type II topoisomerase inhibitors, lipophilic cationic compounds, and DNA intercalating and non intercalating agents. The effects of these drugs on mtDNA metabolism will be discussed and potential mechanisms underlying their depletion of mtDNA presented.

The 6-kb mtDNA of Plasmodium falciparum is thought to replicate by a recombination-dependent mechanism generating large complex branched structures. For technical reasons, including shearing caused by DNA extraction methods, a meaningful quantitative comparison of large complex mtDNA forms has not been feasible. With the use of pulse-field gel electrophoresis, which minimizes any loss or shearing of DNA, we were able to identify an unusually slow migrating population of mtDNA that was resolved from the 6-23-kb population of linear concatemers. Levels of this slow-migrating population of mtDNA were highest during early schizont stage, suggesting that these forms represent replication intermediates. This approach provides a convenient means to monitor the presence of large mtDNA structures in P. falciparum.

Treatment of cultured human cervical carcinoma cells with the anticancer drug dequalinium (DEQ) was found to cause a delayed inhibition of cell growth. This inhibition was preceded by a loss of mitochondrial DNA (mtDNA), a decrease in cytochrome c oxidase activity, and an increase in the level of lactate, indicating that growth inhibition was due to the loss of mtDNA-encoded functions. There was a progressive two-fold loss of mtDNA following each cell division in the presence of DEQ, suggesting that this drug was acting by inhibiting some aspect of mtDNA synthesis. Furthermore, cells became resistant to the growth inhibitory and cytotoxic affects of DEQ when they were grown under conditions that bypassed the need for mtDNA-encoded functions. Resistance was not associated with significant changes in drug accumulation. These results suggest that the DEQ-induced depletion of mtDNA plays an important role in drug cytotoxicity., (Copyright 1998 Academic Press.)

Purpose: Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector., Methods: The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used., Results: Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin., Conclusions: Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.

The topoisomerase II-specific inhibitors VP-16 and ciprofloxacin were used to investigate the presence of topoisomerase II activities associated with nuclear and 35-kb plastid DNAs of the malarial parasite Plasmodium falciparum. The eukaryotic topoisomerase II inhibitor VP-16 induced cleavage of both nuclear and 35-kb parasite DNAs. In contrast, ciprofloxacin, a fluoroquinolone drug known to act on the bacterial type II topoisomerase DNA gyrase, only induced cleavage of the Plasmodial 35-kb DNA. Drug-induced cleavage resulted in the protection of the 5'- but not 3'- ends of the cleaved nuclear and 35-kb DNAs from exonuclease digestion, suggesting that the 5'-ends of the broken DNA were protein-linked, a property reminiscent of DNA cleavage mediated by topoisomerase II enzymes. Furthermore, DNA cleavage induced by both VP-16 and ciprofloxacin was heat-reversible. This is the first evidence that P. falciparum contains two distinct topoisomerase II activities that are molecular targets for chemotherapeutic agents.

We have previously shown that 4-quinolone drugs cause a selective loss of mitochondrial DNA (mtDNA) from mouse L1210 leukemia cells. The loss in mtDNA was associated with a delayed loss in mitochondrial function. Here, we report that the 4-quinolone drug ciprofloxacin is cytotoxic to a variety of cultured mammalian cell lines at concentrations that deplete cells of mtDNA. The IC50 values for ciprofloxacin varied from 40 to 80 micrograms/ml depending on the cell line tested. Cytotoxicity required continuous exposure of cells to drug for 2-4 days, which corresponded to approximately three or four cell doublings. Shorter times of drug exposure did not cause significant cytotoxicity. In addition, cells became drug resistant when they were grown under conditions that bypassed the need for mitochondrial respiration. Resistance was not due to a decrease in cellular drug accumulation, suggesting that ciprofloxacin cytotoxicity is caused by the loss of mtDNA-encoded functions. Analysis of mtDNA from ciprofloxacin-treated cells revealed the presence of site-specific, double-stranded DNA breaks. Furthermore, exonuclease protection studies indicated that the 5'-, but not the 3'-, ends of the drug-induced DNA breaks were tightly associated with protein. These results suggest that ciprofloxacin may be causing cytotoxicity by interfering with a mitochondrial topoisomerase II-like activity, resulting in a loss of mtDNA.

Objective: To evaluate the effectiveness of extended duration clomiphene citrate (CC) (100 mg for 10 days) as an alternative to complex ovulation induction strategies for women who fail to ovulate despite standard incremental doses of CC of > or = 150 mg for 5 days., Design: Retrospective case series., Setting: University-based infertility practice., Patient(s): Thirty women with CC-resistant World Health Organization group II ovulatory disorders., Intervention(s): At least one cycle of 100 mg CC from days 3 to 12., Result(s): Fourteen patients (47%) ovulated during 31 of their 48 cycles (65%). Five women (17%) conceived a total of seven singleton pregnancies, including five term deliveries and two spontaneous abortions. Weight, body mass index, and the presence of hyperandrogenism did not predict responsiveness to the extended duration CC. Side effects were similar to those reported during standard CC treatment., Conclusion(s): An extended 10-day course of CC provides a simple, noninvasive, and inexpensive alternative for a subset of women with ovulatory disorders that are refractory to standard CC treatment.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Previous in vivo studies have identified a prominent 4'-demethylepipodophyllotoxin-9-(4,6-O-thionylidine-beta-D-g lucopyranoside) (VM-26)-induced double-stranded topoisomerase II cleavage site at approximately +80 relative to the start of Drosophila hsp70 transcription (Kroeger, P. E., and Rowe, T. C. (1992) Biochemistry 31, 2492-2502). Topoisomerase II binding at this site correlated with the repression of hsp70 transcription suggesting that this protein-DNA interaction was important in the regulation of hsp70 gene expression. In this paper, we investigated the interaction of purified Drosophila topoisomerase II with a 271-base pair DNA fragment containing the +80 region of the hsp70 gene using the topoisomerase II-specific inhibitor VM-26. VM-26-induced topoisomerase II cleavage of the hsp70 DNA resulted in a major 4-base staggered double-stranded break at +84. In the absence of ATP the +84 site was the only significant VM-26-induced cleavage site. Addition of ATP to the reaction resulted in a stimulation of topoisomerase cleavage throughout the 271-base pair DNA fragment. Deletion analyses determined that approximately 15 to 25 bp of flanking sequence were required for efficient cleavage at most topoisomerase II sites within the hsp70 DNA. However, in the case of the +84 site, topoisomerase cleavage still occurred even when this site was split in half by the restriction enzyme PstI. Topoisomerase II cleavage of both "half-site" DNA molecules occurred at the correct positions on the 4-base single-stranded DNA overhangs generated by PstI. Cleavage was reversible indicating that topoisomerase II could reseal the single-stranded DNA break formed in each half-site substrate. Denaturation of the half-site molecules abolished topoisomerase II cleavage suggesting that cleavage required the duplex region adjacent to the single-stranded cleavage site. Identification of this unusual half-site substrate provides additional evidence that double-stranded cleavage of DNA by topoisomerase II occurs via two sequential single-stranded breaks.

The 4-quinolone antibiotics nalidixic acid and ciprofloxacin are potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout. However, there was a 2- to 4-day lag before the growth rate returned to normal levels. This was preceded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA.

Drug resistance to inhibitors of DNA topoisomerase II can result from qualitative or quantitative alterations in the target enzyme, topoisomerase II, or from perturbations in drug transport that may or may not involve P-glycoprotein. In the present study, a drug-resistant Chinese hamster ovary cell line, SMR16, was selected in the presence of an epipodophyllotoxin (VP-16) and was found to be cross-resistant to all classes of topoisomerase II inhibitors (3-35-fold). The 3-fold level of resistance of these cells to vincristine is likely due to diminished uptake of this drug, and this is not mediated by overexpression of P-glycoprotein. No alteration in transport of VP-16 was observed. Immunoblotting with several polyclonal anti-topoisomerase II antibodies demonstrated that the resistant cells contain approximately two-thirds of the parental enzyme amount. The topoisomerase II catalytic activity present in 0.35 M NaCl nuclear extracts paralleled this decrease. VP-16- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA damage, mediated by topoisomerase II, was found to be decreased 10-12-fold in both intact SMR16 cells and nuclei isolated from these cells, when measured by alkaline filter elution. However, the VP-16-induced DNA cleavage activity present in 0.35 M NaCl nuclear extracts of the resistant cells was attenuated only 2-fold, relative to wild-type cells. Homogeneous preparations of the enzyme obtained from resistant cells demonstrated the same cleavage and catalytic activity as purified wild-type topoisomerase II. Analysis by pulse-field gel electrophoresis of the DNA isolated from VM-26- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-treated sensitive and resistant cells demonstrated significantly less conversion of SMR16 chromosomal DNA into 50-150-kilobase DNA fragments. Chinese hamster ovary SMR16 cells are apparently resistant to topoisomerase II poisons because the topoisomerase II that defines the DNA topological domains is either decreased in amount or insensitive to drug action.

Evaluation of 304 infertile couples with at least one abnormal semen analysis (sperm density < 20 x 10(6)/ml and/or motility < 50%) and no apparent female factors was performed in a multicentre prospective cohort study. In 73 cases therapeutic donor insemination was performed (TDI group) with a resulting pregnancy rate of 48%. The remaining 231 couples (non-TDI group) had an overall pregnancy rate of 25%. The TDI group had a shorter duration of infertility. The ages of both partners were comparable in TDI and non-TDI groups. In the non-TDI group, univariate analysis resulted in identification of six clinical variables associated with a change in pregnancy rates. The strongest association was noted for length of infertility. There was a weaker association for semen volume, concentration of leukocytes in semen, history of pregnancy in the female partner and laparoscopy. Multiple variable analysis of data from the non-TDI group revealed that independent predictors of pregnancy were 'duration of infertility' and 'history of pregnancy in the female partner'. The multiple variable modelling suggested that (i) an increase in the length of infertility by 1 month prolongs the time to pregnancy by an additional 1.6% (95% confidence interval: 1.5-1.7%); and (ii) a history of past pregnancy in the female partner reduces the time of pregnancy by 51% (95% confidence interval: 47-56%).

We have compared topoisomerase I and II cleavage sites on the actin 5C and 57A genes and the hsp70 genes in Drosophila Kc cells using the inhibitors camptothecin (topoisomerase I specific) and VM-26 (topoisomerase II specific) to assess the role of these enzymes in transcriptional regulation. Topoisomerase I cleavage sites were localized to the transcribed regions of the actin 5C and hsp70 genes and were present only when these genes were active. The actin 57A gene, shown previously to be inactive in Kc cells, had no detectable topoisomerase I cleavage sites. In contrast to topoisomerase I, topoisomerase II cleavage sites could be detected on transcriptionally active and inactive actin and hsp70 DNA sequences. Topoisomerase II cleavage sites on the inactive hsp70 gene were primarily localized to the very 5' end of the transcribed region of the gene. However, upon heat-induced activation of hsp70 transcription, topoisomerase II cleavage rapidly shifted from the 5' to the 3' end of the gene. Then, during the shutdown of hsp70 expression, there was a gradual reappearance of topoisomerase II cleavage at the 5' end of the gene that temporally correlated with the repression of hsp70 transcription. There was a similar preferential association of topoisomerase II with the 5' ends of transcriptionally repressed actin 5C and 57A genes. These results demonstrate that there are marked differences in how topoisomerases I and II interact with transcriptionally active and inactive regions of chromatin. In addition, we have identified an unusual type of topoisomerase II binding site that is preferentially associated with the 5' ends of inactive hsp70 and actin genes, suggesting that this enzyme may facilitate changes in chromatin structure that are associated with repression of gene transcription.

Our objective was to determine the effect of treatment on the likelihood of pregnancy among couples with unexplained infertility. We used a nonrandomized, prospective, multicentered cohort analytic study, with mean follow-up time of 14.5 months (range, 0.5-46 months). The subjects were 470 couples who attended infertility clinics affiliated with medical schools in Canada, in whom no abnormality was found after investigation. They were drawn from a total of 2,106 couples registered from April 1, 1984 to March 31, 1987. Of these, 130 couples were selected for treatment at the discretion of the care givers; 340 couples were not treated. Selection for treatment resulted in imbalance between the groups: the treated couples had a longer mean duration of infertility (48 vs. 36 months), and were more likely to have had a laparoscopy as part of the investigation (72% vs. 48%). No specific protocol of treatment was used. Treatments most commonly used were clomiphene (87); gonadotropins (31); intrauterine insemination (20); IVF or GIFT (16); bromocriptine (12); 43 couples had two treatments, and 11 had three treatments. The only important determinants of treatment (logistic regression) were time under observation and laparoscopy status. Duration of infertility was only a minor determinant of treatment. Crude, unadjusted pregnancy rates were 25% for the treated group and 34% for the untreated group. The early occurrence of pregnancy in the untreated couples accounted for much of this difference. After adjustment for baseline differences between the groups and times to and under treatment with proportional hazards analysis, the cumulative probability of pregnancy is 2.0 (95% CI 1.3 to 3.1) times as high with treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The phosphorylation of the nuclear enzyme DNA topoisomerase II was characterized from HeLa human cervical carcinoma cells labeled with 32Pi. Analysis of topoisomerase II immunoprecipitates from 32P-labeled HeLa cells indicated that phosphorylation of the enzyme occurred at serine residues. Incorporation of 32P into topoisomerase II was not due to other types of phosphomodifications such as poly(ADP-ribosylation) or covalent interactions of the enzyme with nucleic acids. The stability of topoisomerase II protein and topoisomerase II phosphorylation was also investigated in HeLa cells. Topoisomerase II protein was relatively stable, having a half-life of approximately 27 h. Phosphorylation of HeLa topoisomerase II was also remarkably stable with a T1/2 of 17 h.

Induction of transient thermotolerance by heat or other cytotoxic stressors has been reported to confer a moderate degree of drug resistance to tumor cells in vitro. In this study, a genetically stable, heat-resistant mouse B16 melanoma variant (W-H75) was tested for its sensitivity to various cytotoxic and antiproliferative agents. The heat-resistant W-H75 cells displayed a moderate two- to threefold resistance to doxorubicin, VP-16, VM-26, colchicine, cis-dichlorodiammineplatinum(II), HgCl2, and CdCl2. Marginal resistance to 4'(9-acridinylamino)methanesulfon-m-anisidide vinblastine, 1,3-bis(2-chloroethyl)-1-nitro-sourea, and NaAsO2 was observed, while no difference in sensitivity to the anticancer drugs, actinomycin D and camptothecin, was observed. Although W-H75 cells were generally more resistant than the parental cells to most of the agents that were tested, they were collaterally sensitive to the antimetabolites methotrexate and 6-mercaptopurine. Resistance of the W-H75 cells to epipodophyllotoxins and anthracyclines was not due to differences in steady-state drug accumulation. For the epipodophyllotoxin VP-16, resistance may be related to a relative decrease in the number of drug-induced DNA strand breaks in W-H75 cells. However, no difference in DNA strand breakage was observed between W-H75 and parental cells which were treated with doxorubicin, suggesting that resistance to this drug occurred by a different mechanism. The possible involvement of glutathione and glutathione S-transferase in resistance was also investigated. The glutathione content in W-H75 cells was 35% higher than that in the parental line. However, glutathione S-transferase activity appeared to be identical in both cell lines. Two other heat-resistant B16 melanoma variants, B-H103 and R-H92, were also tested for sensitivity to doxorubicin and VP-16. In contrast to the W-H75 cells, these two heat-resistant variants were hypersensitive to doxorubicin. The B-H103 cells were also hypersensitive to VP-16. This study suggests that selection for cellular resistance to heat may result in cells that have an altered sensitivity to drugs.

The lower eukaryote Ustilago maydis contains a topoisomerase that removes supercoils from negative and positive superhelical DNA. The enzyme may be a multimeric protein or an aggregate of polypeptides with a native molecular weight of 270,000 as estimated by gel filtration. No cofactors are required by the enzyme, but activity is enhanced by Mg2+. Dependence of activity upon enzyme concentration is not linear. Below a threshold level where topoisomerase cannot ordinarily be detected, addition of H1 histone sharply stimulates activity. ATP and a number of structural analogues inhibit the enzyme.

An endonuclease active upon single-stranded DNA has been extensively purified from Ustilago maydis. The native form of the enzyme appears to be a single polypeptide chain of 55,000 daltons. The enzyme does not require addition of divalent cations for activity, but is stimulated severalfold by the transition metals Co2+, Mn2+, and Zn2+. It is strongly inhibited by ethylenediaminetetraacetic acid and 2-mercaptoethanol. The products of reaction are mononucleotides and small oligonucleotides bearing a 5'-phosphomonoester group. Superhelical DNA is cleaved by the enzyme to an open circular form, then cut to form a linear molecule. Relaxed closed circular duplex DNA is resistant to cleavage. Heteroduplex relaxed circular DNA molecules containing nonhomology or damage in only one strand were constructed and tested as substrates for the enzyme. The rate of enzymatic cleavage was found to be high relative to a control when molecules containing depurinated, deaminated, or radiation damaged sites were tested. Molecules containing a single mismatched base pair were not cleaved any faster than a completely base-paired control, unless reactions were carried out under conditions likely to introduce additional structural distortions in the DNA. Linear duplex DNA exposed to the enzyme is progressively shortened by digestion from both 5' and 3' termini.

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.

A nuclease highly active on single-stranded DNA has been purified 11,200-fold from Ustilago maydis. The enzyme has a sedimentation coefficient of 4.3 S and a Stokes radius of 36 A. The native form of the enzyme appears to be a single polypeptide chain of 68,000 daltons. The enzyme is fully active in the presence of chelating agents but is strongly inhibited by nucleotides. Maximal activity is seen at pH 6. The enzyme hydrolyzes linear DNA in an exonucleotytic fashion from the 5' end liberating 3'-mononucleotides and small oligonucleotides. Degradation of DNA takes place through a distributive mode rather than a processive mode. The enzyme is also active on both single-stranded and duplex circular DNA, dephosphorylates 5'-nucleotides, and hydrolyzes RNA.

Similar to its inhibitory effect on mammalian DNA topoisomerase II, the cytotoxic drug VM26 (teniposide) also interferes with the breakage-reunion reaction of Drosophila melanogaster DNA topoisomerase II. VM26 induces topoisomerase II-mediated DNA breakage in vitro and in cultured D. melanogaster cells presumably by stabilizing an enzyme-DNA cleavable complex. The drug-induced DNA breaks on D. melanogaster hsp70 genes were mapped in cultured cells using the indirect end-labeling procedure. Multiple and specific cleavage sites occurred at both the 3' and 5' ends of the hsp70 genes. A number of these cellular topoisomerase II cleavage sites mapped close to the DNase I-hypersensitive regions of the hsp70 genes. The intensities of several topoisomerase II cleavage sites changed significantly on heat shock induction. Treatment of cultured D. melanogaster cells with VM26 at 25 degrees C resulted in the stimulation of transcription of the hsp70 genes. These results suggest that inhibition of DNA topoisomerase II may lead to heat shock transcription.

Topoisomerase I cleavage sites have been mapped in vivo on the Hsp70 heat shock gene of Drosophila melanogaster cells using the drug camptothecin. Topoisomerase I cleavage was only observed when the Hsp70 gene was transcriptionally active. Site-specific single-strand DNA cleavage by topoisomerase I was confined to the transcribed region of the Hsp70 gene and occurred on both the transcribed and nontranscribed DNA strands. A number of the single-strand breaks on the complementary DNA strands occurred in close proximity giving rise to double-stranded DNA breaks. Inhibition of heat-induced Hsp70 transcription by either Actinomycin D (Act D) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibited topoisomerase I cleavage except at the 5' and to a lesser extent the 3' end of the gene. Camptothecin (100 microM) inhibited transcription of the Hsp70 gene greater than 95%. These results suggest that topoisomerase I is intimately associated with and has an integral part in Hsp70 gene transcription.

Adriamycin (doxorubicin), a potent antitumor drug in clinical use, interacts with nucleic acids and cell membranes, but the molecular basis for its antitumor activity is unknown. Similar to a number of intercalative antitumor drugs and nonintercalative epipodophyllotoxins (VP-16 and VM-26), adriamycin has been shown to induce single- and double-strand breaks in DNA. These strand breaks are unusual because a covalently bound protein appears to be associated with each broken phosphodiester bond. In studies in vitro, mammalian DNA topoisomerase II mediates DNA damage by adriamycin and other related antitumor drugs.

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.

Tubal ligation has become the second most popular method of contraception in Canada, after oral contraception. Refinement of techniques has resulted in sterilization procedures which have minimal potential for failure and high potential for reversibility. Laparoscopic and minilaparatomy techniques allow outpatient "Band-Aid" sterilizations with less risk of complications than more destructive procedures. Laparoscopic application of tubal clips or rings is highly effective, with minimal tubal destruction. Tubal ligation following a pregnancy is more often regretted than is interval sterilization. The search continues for a satisfactory transcervical sterilization procedure.

Antitumor drugs from many chemical classes have been shown to induce protein-linked DNA breaks in cultured mammalian cells and in vitro in the presence of purified mammalian DNA topoisomerase II. The possibility that mammalian DNA topoisomerase II is an intracellular target which mediates drug-induced DNA breaks is supported by the following studies using 4'-(9-acridinylamino)methane-sulfon-m-anisidide (m-AMSA): (a) a single m-AMSA-dependent DNA cleavage activity copurified with calf thymus DNA topoisomerase II activity at all chromatographic steps of the enzyme purification; (b) m-AMSA-induced DNA cleavage by this purified activity resulted in the covalent attachment of protein to the 5'-ends of the DNA via a tyrosyl phosphate bond. This covalently linked protein has the same reduced molecular weight as purified calf thymus DNA topoisomerase II. The possibility that topoisomerase II-mediated DNA breaks may be responsible for cytotoxicity has also been investigated using a number of m-AMSA-related acridines. The level of topoisomerase II-mediated DNA breaks in vitro strongly correlates with the level of protein-linked DNA breaks in cultured cells and drug-induced cytotoxicity. These results suggest that mammalian DNA topoisomerase II may be a cytotoxic target of antitumor acridines.

Among 2,106 couples registered in 12 Canadian infertility clinics, 470 (22.3%) were classed as unexplained infertility after a uniform evaluation of the male ejaculate, ovulation, and tubal patency. The unexplained group included more older female partners; 44% were over 30 years of age at registration in the participating clinics, compared with 36% in other infertility diagnostic groups. The mean duration of infertility was 40.1 months, and the cumulative pregnancy rate was 36.6 +/- 2.9% at 2 years after registration. When the variables were examined with the use of proportional hazards analysis, each additional month of duration of infertility reduced the expected prognosis by 2%, and a history of pregnancy in the partnership improved the prognosis by 80%. Among couples with 3 years or more duration of infertility (cumulative pregnancy rate, 27.5 +/- 3.9%), an additional year in the age of the female partner when conception was first attempted (mean, 26.8 years) reduces the prognosis by 9%.

We have purified an acid-soluble DNA endonuclease, termed nuclease gamma, from Ustilago cell extracts. The enzyme is nearly homogeneous, purified 1700-fold. The protein appears to be globular with a molecular weight in the range 17,000 to 21,000. It requires a divalent cation and is optimally active at slightly alkaline pH. The enzyme prefers duplex DNA as substrate but will slowly cleave single-stranded DNA. Cleavage of covalently closed duplex DNA is unaltered by changes in superhelix density. Divalent cations direct the mode by which the enzyme cleaves duplex DNA. When Mg2+ or Ca2+ is added, the enzyme nicks one strand of the duplex. When Mn2+, Co2+, or Zn2+ is added, the enzyme can introduce double strand breaks. Oligonucleotides terminated with 5'-phosphoryl and 3'-hydroxyl groups are the products of hydrolysis. DNA fragments generated can be religated to linear pBR322 DNA with completely base-paired ends.

One hundred seventeen patients with amenorrhea and galactorrhea or hyperprolactinemia were evaluated with regard to antecedent factors, results of investigations, and management. Full details of the outcome of prolonged follow-up were available for 104 patients. Patients who developed amenorrhea-galactorrhea after withdrawal of oral contraceptives or postpartum had a lower incidence of pituitary adenomas than did those who developed amenorrhea-galactorrhea spontaneously. Six of a total of 40 tumors were detected only during the follow-up period. This study suggests that patients with spontaneous amenorrhea-galactorrhea have a greater risk of developing a detectable pituitary adenoma than do those with postpill or postpartum symptoms. However, patients with a microadenoma are more likely to have had postpill onset of hyperprolactinemia. Plasma prolactin (PRL) in patients with postpill amenorrhea-galactorrhea increased in proportion to the duration of oral contraceptive use.

Highly purified topoisomerase from Ustilago breaks single-stranded DNA, forming a complex with protein covalently bound to the DNA. Methods used to detect the complexes include a nitrocellulose filter assay, electrophoresis of the DNA-protein complex in agarose gels containing alkali, and isolation of the complex after removal of all but a small oligonucleotide fragment bound to the protein. The linkage of the Ustilago topoisomerase is to the 3' end of the broken strand of DNA. The DNA-protein complex formed is through a phosphodiester bond to tyrosine.

The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agents appear to be mediated by DNA topoisomerase II. We have purified topoisomerase II to homogeneity both from an epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, and from the wild-type parental cell line. Immunoblots demonstrate similar topoisomerase II content in these two cell lines. The purified enzymes are dissimilar in that DNA cleavage by VpmR-5 topoisomerase II is not stimulated by VP-16 or m-AMSA. Furthermore, the VpmR-5 enzyme is unstable at 37 degrees C. Thus, the drug resistance of VpmR-5 cells appears to result from the presence of an altered topoisomerase II in these cells. Purified topoisomerase II from VPMR-5 and wild-type cells has the same monomeric molecular mass as well as equivalent catalytic activity with respect to decatenation of kinetoplast DNA. Etoposide (VP-16) inhibits the activity of both enzymes. Noncovalent DNA-enzyme complex formation, assayed by nitrocellulose filter binding, is also similar, as is protection from salt dissociation of this complex by ATP and VP-16. The data suggest a model in which the drug-resistant cell line, VpmR-5, has religation activity which is less affected by drug than that of the wild-type cells. Drug effect on DNA religation and catalytic activity are dissociated mechanistically. In addition, under certain circumstances, the "cleavable complex" observed following denaturation of a drug-stabilized DNA-enzyme complex may not adequately reflect the nature of the antecedent lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Seven women with stress-induced amenorrhea were challenged with metoclopramide, 10 mg intravenously, before and at the end of a course of clomiphene. Initial testing with luteinizing hormone releasing hormone demonstrated that all subjects had the capacity to release luteinizing hormone (LH), but in response to metoclopramide there was no increase in the levels of LH. This lack of response did not change after 5 days of clomiphene, although basal levels of LH and estradiol increased significantly. The pattern of response of prolactin to metoclopramide did not change after clomiphene. These results suggest that there is no significant dopamine-mediated inhibition of release of luteinizing hormone releasing hormone in women with stress-induced amenorrhea. The administration of clomiphene to these women does not appear primarily to alter hypothalamic dopaminergic activity.

The effect of antitumor epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), on chromosomal DNA in mammalian cells was studied using SV40 virus-infected monkey cells as a model system. Treatment of SV40 virus-infected monkey cells with these drugs results in DNA breaks on intracellular SV40 DNA. The broken DNA strands are sensitive to phenol extraction, suggesting that they are associated with tightly linked protein(s). Several pieces of evidence suggest that DNA topoisomerase II is covalently linked to the broken SV40 DNA strands following drug treatment. ovobiocin, an inhibitor of topoisomerase II, blocks the epipodophyllotoxin-induced SV40 DNA breaks in vivo and in vitro. Epipodophyllotoxin-induced cleavage sites on intracellular SV40 DNA are strikingly similar to those produced on purified SV40 DNA by purified calf thymus DNA topoisomerase II. The protein-linked SV40 DNA is specifically immunoprecipitated by antisera against topoisomerase II. We thus conclude that epipodophyllotoxins induce chromosomal DNA breakage via DNA topoisomerase II. The physiological effects of epipodophyllotoxins on cell death, chromosomal DNA breakage, sister chromatid exchanges, and chromosomal aberrations may be the consequence of drug interaction with DNA topoisomerase II. Our present results are also consistent with the proposal that epipodophyllotoxins interfere with the breakage-reunion reaction of DNA topoisomerase II by stabilizing an enzyme-DNA complex in its putative cleavable state.

Camptothecin is a cytotoxic drug which inhibits cellular nucleic acid synthesis. Associated with this inhibition is the induction of protein-linked DNA strand breaks. Recent studies have demonstrated that camptothecin interferes with the DNA breakage and rejoining activity of the enzyme DNA topoisomerase I and stabilizes a cleavable complex between this enzyme and DNA. Treatment of this complex with a protein denaturant results in DNA strand breaks and the covalent attachment of topoisomerase to the 3'-end of the DNA breaks. In this paper we have mapped the camptothecin-induced DNA breaks on the hsp 70 heat-shock gene in cultured Drosophila cells. Drug-induced breaks are present primarily within the coding region of this gene and occur only when transcription of this gene is activated by heat. Camptothecin (20 microM) was also observed to inhibit heat-induced hsp 70 transcription greater than 70%. The possible role of topoisomerase I in hsp 70 heat-shock gene transcription is discussed.

A low-dose "nonsuperovulation" scheme of ovulation induction (schedule 1: 15 treatment cycles in 13 women) was compared to a high-dose, individually adjusted "controlled-superovulation" scheme of human menopausal gonadotropin administration (schedule 2: 18 treatment cycles in 17 women, with four women also having been treated under schedule 1). In schedule 2 more human menopausal gonadotropin was employed, 17 beta-estradiol plasma levels were higher, and more ova were retrieved per treatment cycle (p less than 0.01) with a higher rate of mature ova (p = 0.001). Also, under this schedule all treatment cycles resulted in successful retrieval of ova, whereas under schedule 1, four cycles (27%) were abandoned before laparoscopy because the follicles had ovulated (p = 0.03). Furthermore, the proportion of embryos cleaving to the two- to four-cell stage were higher under schedule 2 (38 of 53, or 72%) as compared to schedule 1 (13 of 25, or 52%), but this was not statistically significant (p = 0.07). Two pregnancies were conceived under schedule 2 giving a rate of 11% per treatment cycle. It was concluded that schedule 2, the individually adjusted controlled-superovulation scheme of human menopausal gonadotropin administration, was the superior technique of ovulation induction in an in vitro fertilization program.

The antitumor drug 4'-(9-acridinylamino)methanesulfon-m-anisidide which stimulates the cleavable complex formation between mammalian DNA topoisomerase II and DNA also stimulates the cleavable complex formation between bacteriophage T4-induced DNA topoisomerase and DNA. In the presence of 4'-(9-acridinylamino)methanesulfon-m-anisidide, T4 DNA topoisomerase and DNA form a "cleavable complex" which is characterized by its sensitivity to protein-denaturant treatment. Upon protein-denaturant treatment, the phosphodiester bond of DNA is cleaved, and the gene 52 protein subunit of the topoisomerase becomes covalently linked to the 5'-end of the broken DNA. The covalent protein-DNA linkage has been determined by both paper electrophoresis and thin layer chromatography to be tyrosyl phosphate.

The antitumor drug, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide), is known to interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by blocking the enzyme-DNA complex in its putative cleavable state. Treatment of SV40 virus infected monkey cells with m-AMSA resulted in both single- and double-stranded breaks on SV40 viral chromatin. These strand breaks are unusual because they are covalently associated with protein. Immunoprecipitation results suggest that the covalently linked protein is DNA topoisomerase II. These results are consistent with the proposal that the drug action in vivo involves the stabilization of a cleavable complex between topoisomerase II and DNA in chromatin. Mapping of these double-stranded breaks on SV40 viral DNA revealed multiple topoisomerase II cleavage sites. A major topoisomerase II cleavage site was preferentially induced during late infection and was mapped in the DNAase I hypersensitive region of SV40 chromatin.