Plasma constituents and mortality in rat pups given chronic insulin via injection, pellet, or osmotic minipump

Two studies compared the glucose responses of 9-day-old rats given subcutaneous insulin, either continuously or via daily injection, for 10 days. In Experiment 1, implanted pellets released a total of 0, 1.9, or 5.7 U insulin/kg the first 24 h. Injected doses were larger, 0 or 8 U/kg. Injections caused no deaths, but insulin-releasing pellets caused high mortality within 24 h. Pups surviving the pellets were normoglycemic by treatment day 8. In Experiment 2, pups received 0.184 U of insulin daily, approximately 8 U/kg at 9 days, via either injection or osmotic minipump. All pups survived. Injected pups were hypoglycemic 2 h postinjection through treatment day 10, whereas pups with insulin minipumps were normoglycemic by day 5. Insulin injections, but not minipumps, lowered plasma triglycerides on day 10. To examine age differences in response to insulin, additional pups and adults received daily injections of 0 or 8 U/kg for 10 days. All survived. Insulin lowered plasma glucose more in pups than in adults and reduced triglycerides in pups but not in adults. The rapid development of normoglycemia in pups with insulin minipumps, compared with pups injected daily with the same dose, suggests that continuous early insulin may produce insulin resistance.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4 dinitro-1-fluorobenzene (DNFB) was induced in BALB/c mice by DNFB skin application. Development of skin CH was suppressed by exposure of the animals after sensitization to the cancer chemotherapeutic drugs cyclophosphamide (CY), sodium methotrexate (MTX), and 5-fluorouracil (5FU). Unresponsiveness to DNFB was also induced in parallel experiments by a single intravenous injection of dinitrobenzenesulfonate (DNBS), either before or concomitant with sensitization. Potentiation of CH skin reactivity was achieved by administration of CY prior to sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of animals exposed to CY, MTX, or 5FU to respond to DNFB sensitization. The agent did not impair the potentiation of CH skin reactivity that could be effected by administration of CY prior to sensitization.MER treatment was not effective in reversing hapten-induced (DNBS) tolerance in mice.These findings favor the assumption that MER, under the conditions tested, stimulates the function of positively reacting T cells and exerts no enhancing or protective action on suppressor T cells.     

Acute phase protein response and polymorphonuclear leukocyte cathepsin g release after slow interleukin-1 stimulation in the rat

In this work we have studied the acute phase protein response and degranulation of polymorphonuclear leukocytes in vivo in the rat after a slow interleukin-1beta stimulation. A total dose of 1 mug, 2 mug, 4 mug and 0 mug (controls with only vehicle) of interleukin-1beta was released from osmotic minipumps over a period of 7 days. The pumps were implanted subcutaneously. A cystic formation was formed around the pumps that contained interleukin-1beta whereas no tissue reaction was seen around pumps containing only vehicle. Besides flbroblasts the cyst wall contained numerous polymorphonuclear leukocytes which were positively stained for cathespin G. alpha(2)-macroglobulin, alpha(1)-inhtbitor-3, alpha(1)-proteinase inhibitor, albumin and C3 were measured by electroimmunoassay and all showed plasma concentration patterns that were dose-dependent to the amount of interleuktn-1beta released. Fibrinogen in plasma was elevated in the control group but showed decreased plasma values with higher doses of interleukin-1beta released. All animals showed increased plasma levels of cathespin G but the lowest levels for cathespin G were seen for the highest interleukin-1beta dose released. It was clearly seen that a slow continuous release of interleukin-1beta in vivo caused an inflammatory reaction. Plasma levels for the proteins analysed all showed a similar pattern, namely an initial increase or decrease of plasma concentration followed by a tendency to normalization of plasma values. It was concluded that a long-term interleukin-1beta release could not sustain the acute phase protein response elicited by the initial interleukin-1beta release.     

Morphology of the pancreas during intra-aortal administration of 5-fluorouracil

The results of the studies of morphological changes in the pancreas during intraaortal injections of various 5-fluorouracil doses to 44 dogs are reported. It was experimentally established that intraaortal injection of 10 mg 5-fluorouracil per kg of mass for 3 days caused the delay in albumin-synthesizing organella function, without damaging organellas as such. Administration of a cytostatic in a dose of 15 mg per kg of mass did not only cause a drastic decline in the synthesizing activity of cells in acinar parenchyma, but also induced intracellular lesions.     

Correlation between decreased sensitivity of the Daudi lymphoma cells to VP-16-induced apoptosis and deficiency in DNAS1L3 expression

A functional relationship between the apoptotic endonuclease DNAS1L3 and the chemotherapeutic drug VP-16 was established. The lymphoma cell line, Daudi, exhibited a significant resistance to VP-16 treatment in comparison to the lymphoma/leukemia cell line, U-937. While U-937 cells degraded their DNA into internucleosomal fragments, Daudi cells failed to undergo such fragmentation in response to the drug. Activation of both caspase-3 and DNA fragmentation factor was not sufficient to trigger internucleosomal DNA fragmentation in Daudi cells. No correlation was found between expression levels of topoisomerase-II, Pgp, Bcl-2, Bax, or Bad and decreased sensitivity of Daudi cells to VP-16. Daudi cells failed to express DNAS1L3 and ectopic expression of this protein significantly sensitized the cells to VP-16. An enhancement of caspase-3 activity and collapse of mitochondrial membrane potential underlie DNAS1L3-mediated sensitization of Daudi cells to VP-16, which may be a direct result of DNAS1L3-mediated increase in PARP-1-activating DNA breaks after VP-16 treatment. Our results suggest that DNAS1L3 plays an active role in lymphoma cell sensitization to VP-16 and that its deficiency may constitute a novel mechanism of drug resistance in these cells.     

Chronic administration of the antidepressants phenelzine,despiramine, clomipramine,or maprotiline decreases binding to 5-hydroxytryptamine2A receptors without affecting benzodiazepine binding sites in rat brain

Summary 1. The effects of chronic administration of five antidepressant drugs on the benzodiazepine and 5-HT_2A binding sites in the same rat brain were assessed.2. Clomipramine, desipramine, maprotiline, fluoxetine, and phenelzine (all 10 mg/kg/day) were administered s.c. for 21 days by Alzet osmotic minipumps.3. Results showed that none of the drugs changed the density or affinity of benzodiazepine binding sites, yet at the same dose all the drugs with the exception of fluoxetine decreased binding to 5-HT_2A receptors in the same animals.     

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.     

Meiotic stage-dependent induction of chromosome aberrations in Chinese hamster primary oocytes exposed to topoisomerase II inhibitor etoposide

To investigate the chromosomal effects of topoisomerase II (topo-II)-interactive drugs on mammalian primary oocytes, female Chinese hamsters were treated with etoposide (VP-16) at various intervals pre- and post-human chorionic gonadotropin (hCG) injections. Chromosome analysis of oocytes at metaphase II (M II) showed that treatment with VP-16 at 50h pre-hCG had no effect, but the treatments between 24h pre-hCG and 2h post-hCG often caused structural chromosome aberrations. Although treatment at 4h post-hCG had no effect, subsequent treatments at 6 and 8h post-hCG produced a significant increase in structural chromosome aberrations. No effect was found following treatment at 10h post-hCG. The incidence of aneuploidy following exposure to VP-16 was also dependent on the time of hCG injection. Taking the time course of meiotic progression in primary oocytes following hCG injection and pharmacokinetics of VP-16 into consideration, it is likely that meiotic stages from late dictyate to diakinesis are highly sensitive to VP-16, while stages at dictyate and from metaphase I (M I) to telophase I (telo I) are relatively insensitive to the drug. Moreover, the effect of VP-16 on structural chromosome aberrations and aneuploidy was dose-dependent.Chromosome analysis at M I detected a frequent occurrence of structural chromosome aberrations in treated oocytes. This suggests that structural aberrations may be caused by disruption of cleavable complexes during chromosome condensation. Detection of chromosome bridges during anaphase I/telophase I (ana I/telo I) may support the hypothesis that induction of aneuploidy by VP-16 is due to failure in decatenation of recombinant homologous chromosomes.     

The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.     

Valproic acid-induced neural tube defects: reduction by folinic acid in the mouse

Neural tube defects were induced dose-dependently by single injections of the anticonvulsant drug valproic acid (VPA) as sodium salt in mice on gestational day 8. Folinic acid (5-CHO-THF) coadministration by i.p. injection or by a constant rate infusion via osmotic minipumps, implanted s.c., significantly reduced the exencephaly rates using a randomized double-blind experimental procedure. 5-CHO-THF supplementation cut the exencephaly rates into half even at high maternal plasma levels of VPA (p less than 0.005, chi 2-test); resorption rates were not affected. The VPA plasma kinetics were not changed by any of the application regimens of 5-CHO-THF. The investigation of the folate metabolite pattern (determined by HPLC) showed that 5-CHO-THF and 5-methyl-tetrahydrofolic acid (5-CH3-THF) were the main metabolites in untreated mice. After supplementation with 5-CHO-THF, only the concentrations of this folate vitamer were increased in the plasma from 0.3 microgram/ml (normal) to 0.6 or 1.9 micrograms/ml (after injection of 3 x 1 mg/kg or 3 X 4 mg/kg) and to 4.2 micrograms/ml (after infusion via osmotic minipumps). Our results indicate that VPA-induced exencephaly in mice combined with the investigation of the plasma levels of VPA and the different folate metabolites could be an appropriate animal model to study protective effects of folates on the occurrence of neural tube defects.     

The antihypertensive effect of U-0521 (3′,4′-dihydroxy-2-methylpropiophenone)

The synthetic catechol, U-0521, (3′,4′-dihydroxy-2-methylpropiophenone) is a competitive inhibitor of both tyrosine hydroxylase and catechol-O-methyltransferase. Continuous subcutaneous administration of 10 μmoles per day of U-0521 via Alzet osmotic minipumps to adult male Spontaneously Hypertensive Rats (SHR) reduced blood pressure from 160 mmHg to 125 mmHg. This effect occurred within two days, persisted for the two weeks that the pumps were in place, and reversed gradually upon cessation of U-0521 administration. Similar treatment of U-0521 to normotensive Wistar Kyoto rats (WKY) did not result in a similar hypotentensive effect. Subcutaneous administration of the same dose to juvenile SHRs led to a blockade in the expression of hypertension. After the five week treatment period, the blood pressure of the U-0521 treated animals escalated rapidly to match the saline treated controls. The antihypertensive effect of U-0521 on SHRs also occured when the compound was delivered by the oral route at the rate of 50 mg/kg/day.     

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.     

Embryotoxicity of the intercalating agents m-AMSA and o-AMSA and the epipodophyllotoxin VP-16 in postimplantation rat embryos in vitro

The intercalating agent, m-AMSA, and the epipodophyllotoxin, VP-16, both topoisomerase II-reactive anticancer agents, are also embryotoxic agents in rat embryos cultured in vitro. Quantifying the embryotoxic effects of these drugs revealed that the no observed adverse effect level (NOAEL) for m-AMSA is 10 nM, the embryotoxic concentration range is 50-500 nM, and complete lethality is observed at 1 microM. In contrast, the NOAEL for o-AMSA, an inactive isomer of m-AMSA, is 1.0 microM, the embryotoxic concentration range is 10-100 microM, and complete lethality occurs at 200 microM. Based upon the concentrations of drugs required to produce 50% embryotoxicity or 50% malformed embryos, m-AMSA exhibits a 200-500-fold-higher embryotoxicity compared to o-AMSA. VP-16 exhibits a NOAEL of 1.0 microM, an embryotoxic concentration range of 2-5 microM, and complete lethality at 10 microM. Compared to m-AMSA, VP-16 is approximately 10-fold less embryotoxic. At appropriate concentrations, all three drugs were dysmorphogenic resulting in embryos that were characterized by hypoplasia of the prosencephalon with associated microopthalmia and dilation of the rhombencephalon. and dilation of the rhombencephalon. As a prelude to future studies focusing on the mechanism of drug-induced embryotoxicity, we have used established biochemical and immunologic methods to identify and quantify topoisomerase II in rat embryos. In addition, we have demonstrated that the embryo topoisomerase II can be inhibited by both m-AMSA and VP-16. Finally, we have used a human cDNA probe to detect topoisomerase II mRNA in the rat embryo. Thus, the combination of the in vitro whole embryo culture and these biochemical/molecular assays should allow us to explore the role of a specific nuclear target, i.e., topoisomerase II, in the teratogenic effects of some commonly employed chemotherapeutic agents.     

Activation of serotonin (5-HT)1A receptors inhibits amphetamine sensitization in mice

The effects of serotonin (5-HT)1A drugs on the development and expression of sensitization to the locomotor effect of amphetamine (AMPH) were studied in mice. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A agonist, dose-dependently reduced the expression of AMPH (2.5 mg/kg)-induced sensitization. The latter inhibitory effect of 8-OH-DPAT was reversed by (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenyl propamine (WAY 100135), a 5-HT1A antagonist. WAY 100135 given alone did not affect expression of AMPH sensitization. Combined injections of 8-OH-DPAT, but not WAY 100135, with AMPH (2.5 mg/kg) during the development of sensitization, protected against the expression of sensitization to a challenge dose of AMPH (2.5 mg/kg) 3 days after withdrawal. The above inhibitory effect of 8-OH-DPAT on the development of AMPH sensitization was blocked by pretreatment with WAY 100135. The AMPH-induced conditioned locomotion was unaffected by pretreatment with 8-OH-DPAT. These results indicate that 5-HT1A receptors are not involved in AMPH-induced sensitization per-se, whereas their pharmacological activation leads to the inhibition of both the development and the expression of AMPH-induced sensitization.     

Role of prolactin and luteinizing hormone in regulating timing of implantation in the spotted skunk

The western spotted skunk exhibits an obligate delay of implantation lasting 200-220 days. The pituitary is essential for luteal activation. The corpora lutea, in turn, secrete the hormones necessary for blastocyst implantation. Two experiments were designed to determine which pituitary hormones are responsible for increasing luteal activity and induction of implantation. Forty-two pregnant skunks with delayed implanting blastocysts were treated as follows: 13 served as untreated controls, 6 received 0.5 mg prolactin (PRL) daily, and 5 received diluent beginning in January. Four received 1.5 mg bromocriptine (CB-154) daily, 3 received both CB-154 and PRL, 3 received diluent, 5 received a gonadotropin-releasing hormone agonist (GnRHa) dispensed from osmotic minipumps, and 3 received diluent dispensed from osmotic minipumps starting in April. The skunks were subjected to a natural photoperiod. Duration of preimplantation and blood levels of progesterone and luteinizing hormone were measured. PRL significantly (p less than 0.05) shortened and CB-154 significantly (p less than 0.05) prolonged the duration of preimplantation when compared to controls (148 +/- 33.6 vs. 251 +/- 3.2 vs. 199 +/- 5.1 days, respectively). PRL was able to reverse the inhibitory effect of CB-154 when both were administered simultaneously (195 +/- 4.0 vs. 251 +/- 3.2 days). GnRHa had no significant (p greater than 0.05) effect on duration of preimplantation (199 +/- 5.1 days) when compared to controls (203 +/- 3.2 days). These results indicate that PRL is the primary pituitary hormone responsible for increased luteal activity and subsequent blastocyst implantation in the spotted skunk.     

Parallel formation of delayed-hypersensitivity effectors and T-suppressors in the intraperitoneal administration of a massive dose of ram erythrocytes

Injection of 6 X 10(9) sheep red blood cells (SRBC) to mice led to parallel formation, on days 4-5, of delayed hypersensitivity effector cells (the activity was tested in local transfer experiments) and delayed hypersensitivity T-suppressors preventing sensitization of syngeneic recipients. After massive injection of SRBC the activity of spleen suppressors gave 2 peaks: on days 5 and 14. Five days after massive antigen injection only T-cells capable of sorbing on a specific antigen manifested suppressor activity. On day 14 T-cells capable of sorbing on specific antibodies showed a specific activity, whereas T-cells capable of sorbing on a specific antigen retained only part of their activity. The mechanism of delayed hypersensitivity inhibition following massive antigen injection by suppressors obtained by day 5 is reviewed in terms of Germain and Benacerraf's theory postulating that delayed hypersensitivity is regulated by Ly 2+, I-J+ antiidiotypic suppressors capable of sorbing on specific antibodies and formed upon injection of Ly 1+, I-J+, Id+ inductor cells capable of sorbing on a specific antigen.     

Dose response of hepatic and renal DNA synthetic rates to continuous exposure of bromodeoxyuridine (BrdU) via slow-release pellets or osmotic minipumps in male B6C3F1 mice

We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney of B6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (IP) injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one of four BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs of the animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nuclear labeling compared with those receiving a single-pulse injection. No time-related increases in nuclear labeling were detected in hepatocytes or renal tubule cells of mice exposed to BrdU pellets and in the kidneys of mice receiving BrdU minipumps at the 7-day compared with the 4-day time point. In some cases, the labeling indices at 7 days were significantly decreased compared with those at 4 days. In contrast, a time-related increase in nuclear labeling was seen in hepatocytes and Kupffer cells of mice exposed to BrdU minipumps. Therefore, the method used to administer BrdU chronically to the animal appears to play an important role in presenting the true proliferative scenario in cell kinetic studies. Our findings also provide evidence for an effect of BrdU on normal proliferation rates in these tissues.     

Differential recovery of intestine, bone marrow, and thymus of rats with solid tumors following 5-fluorouracil administration.

Time relationships for recovery of several host organs from toxic effects of 5-fluorouracil were determined in ACI rats bearing Morris hepatoma 3924A. A single injection of 150 mg/kg body weight 5-fluorouracil (the LD10) resulted in loss of 90% of the tibial bone marrow, 60% of the intestinal mucosa, and 90% of the thymus as measured by total DNA content of the organs. Organ DNA contents following 150 mg/kg of the drug were minimal on day 3 for intestine and on day 5 for marrow and thymus. A return to pretreatment or higher levels of DNA was observed by day 4 for intestine, day 11 for tibial marrow, and day 19 for thymus. Incorporation of 3H-deoxyuridine into host organ DNA after 150 mg/kg 5-fluorouracil was inhibited 36 hrs for intestine, 3 days for thymus, and 5 days for tibial bone marrow. Inhibition of 3H-deoxyuridine incorporation into DNA was similar for 50, 100, and 150 mg/kg doses both in tumor and in host organs, but recovery of 3H-deoxyuridine incorporation and DNA content of host organs began later with the higher doses of 5-fluorouracil. Maximal incorporation of 3H-deoxyuridine into DNA was observed on day 4 for intestine, day 8 for marrow, and day 9 for thymus after treatment with 150 mg/kg 5-fluorouracil. Animal lethality following the second of two 150 mg/kg injections of 5-fluorouracil was related to the extent of recovery of intestinal mucosa and bone marrow at the time of the second injection. Survival decreased to 0% for normal rats when the interval between injections was 3-4 days, improved at 5 days and was 100% when the interval was 10-11 days.     

Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions

A Ser740 --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2(S740W) in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position -2 and G at position +6 were observed for the top2(S740W) in addition to the previously reported C-1 and G+5 for the wild-type top2. The VP-16-induced top2(S740W) cleavage complexes were also more stable. The most stable sites had strong preference for C-1, whereas the most reversible sites showed no base preference at positions -1 or -2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser740 --> Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.