Treatment of refractory acute leukemia with timed sequential chemotherapy using topotecan followed by etoposide + mitoxantrone (T-EM) and correlation with topoisomerase II levels.

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.

Mitochondrial DNA metabolism targeting drugs.

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.

Analysis of Plasmodium falciparum mitochondrial six-kilobase DNA by pulse-field electrophoresis.

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.

Dequalinium induces a selective depletion of mitochondrial DNA from HeLa human cervical carcinoma cells.

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.

DQAsomes: a novel potential drug and gene delivery system made from Dequalinium.

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.

Topoisomerase II inhibitors induce cleavage of nuclear and 35-kb plastid DNAs in the malarial parasite Plasmodium falciparum.

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.

An extended 10-day course of clomiphene citrate (CC) in women with CC-resistant ovulatory disorders.

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.

Delayed cytotoxicity and cleavage of mitochondrial DNA in ciprofloxacin-treated mammalian cells.

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.

Analysis of expressed sequence tags from Plasmodium falciparum.

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.

Analysis of topoisomerase II-mediated DNA cleavage in the 5'-region of the Drosophila hsp70 gene. Identification of a novel half-site DNA substrate for topoisomerase II cleavage.

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.

Topoisomerase II activity involved in cleaving DNA into topological domains is altered in a multiple drug-resistant Chinese hamster ovary cell line.

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.

4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells.

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.

Prognostic factors in assessment and management of male infertility.

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%).

Analysis of topoisomerase I and II cleavage sites on the Drosophila actin and Hsp70 heat shock genes.

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.

The effect of treatment on pregnancy among couples with unexplained infertility.

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)

Phosphorylation of DNA topoisomerase II in a human tumor cell line.

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.

Drug sensitivity of heat-resistant mouse B16 melanoma variants.

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.

Interaction of topoisomerase 1 with the transcribed region of the Drosophila HSP 70 heat shock gene.

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.

Age of the female partner is a prognostic factor in prolonged unexplained infertility: a multicenter study.

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%.