Effect of taxol and taxotere on gene expression in macrophages: induction of the prostaglandin H synthase-2 isoenzyme

Induction of genes encoding cytokines or other, unidentified proteins may contribute to the pharmacological effects of taxol. We hypothesized that prostaglandin H synthase-2 (PGHS-2) was one of the unidentified genes induced by taxol. Taxol alone or taxol plus IFN-gamma increased PGE2 formation, PGHS-2 protein expression, and PGHS-2 mRNA expression in RAW 264.7 murine macrophages. The kinetics for mRNA induction, protein expression, and catalysis were self-consistent. A selective inhibitor of PGHS-2 blocked PGE2 formation by cells incubated with taxol; a selective inhibitor of PGHS-1 had no effect. A glucocorticoid blocked the induction of mRNA, the expression of PGHS-2 protein, and the formation of PGE2. Neither taxol alone nor taxol plus IFN-gamma altered the expression of the PGHS-1 isoenzyme in RAW 264.7 cells. Taxotere, an analogue that stabilizes microtubules as potently as taxol, did not alter the expression of PGHS-2, implying that its induction in RAW 264.7 murine macrophages did not originate from microtubule stabilization. Taxol and taxotere each induced PGHS-2 expression in human monocytes suspended in 10% human serum. However, human monocytes suspended in 10% bovine serum responded only to LPS, not to taxol or taxotere, implying that they act independently of the LPS-mimetic process that is prominent in mice. Taxol induced PGHS-2 in human and murine monocytes via a p38 mitogen-associated protein kinase pathway. The inclusion of PGHS-2 among the early response genes induced in leukocytes may be relevant to the beneficial and adverse effects encountered during taxol administration.     

In vitro cross-resistance and collateral sensitivity in seven resistant small-cell lung cancer cell lines: preclinical identification of suitable drug partners to taxotere, taxol, topotecan and gemcitabin

The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis[4-[3-methy l-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO) binds to double-stranded DNA (dsDNA) in a sequence selective bisintercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans over two base pairs in the minor groove. The binding of analogs of TOTO in which the linker has been modified is examined. The aim of the study is to utilize the sequence selectivity of the TOTO chromophores to enhance and/or alter the overall selectivity of the binding. One- and two-dimensional 1H-NMR investigations of complexes between TOTO analogs and various dsDNA oligonucleotides are reported. The following analogs were synthesized and used: 1,1'-(4,4,8,8-tetramethyl-4,8-diazadodecamethylene) -bis[4-[3-methyl-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO10), 1,1'-(5,5,9,9-tetramethyl-5,9-diazatridecamethylene)-bis[4-[3-meth yl-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO11), and 1,1'-(6,6,10,10-tetramethyl-6,10-diazapentadecamethylene)-bis[4-[3 -methyl-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO13). The results show that with a longer linker the dyes can bisintercalate into two (5'-CpT-3'):(5'-ApG-3') sites separated by one or two base pairs. Bisintercalation in two such "isolated" binding sites yields non-nearest-neighbor bisintercalation in which the linker spans over more than two base pairs. The investigations also showed that an exact length of the linker is not crucial for the site selectivity since TOTO, TOTO10, and TOTO11 are almost equally suitable in binding selectively to the (5'CTAG-3')2 sequence. Fluorescence measurements show that TOTO10, TOTO11, and TOTO13 have higher fluorescence quantum yields than TOTO when bound to d(CGCTAGCG)2. This indicates that the length of the linker in TOTO may not be the optimum one in terms of using the dye as a fluorescence marker.     

Relationship between microtubule dynamics and lamellipodium formation revealed by direct imaging of microtubules in cells treated with nocodazole or taxol

Microtubules (MTs) contribute to the directional locomotion of many cell types through an unknown mechanism. Previously, we showed that low concentrations (<200 nM) of nocodazole or taxol reduced the rate of locomotion of NRK fibroblasts over 60% without altering MT polymer level [Liao et al., 1995: J. Cell Sci. 108:3473-3483]. In this paper, we directly measured the dynamics of MTs in migrating NRK cells injected with rhodamine tubulin and treated with low concentrations of nocodazole or taxol. Both drug treatments caused statistically significant reductions (approx. twofold) in growth and shortening rates and less dramatic effects on rescue and catastrophe transition frequencies. The percent time MTs were inactive (i.e., paused) increased greater than twofold in nocodazole- and taxol-treated cells, while the percent time growing was substantially reduced. Three parameters of MT dynamics were linearly related to the rates of locomotion determined previously: rate of shortening, percent time pausing and percent time growing. The number of MTs that came within 1 microm of the leading edge was reduced in drug-treated cells, suggesting that reduced MT dynamics may affect actin arrays necessary for cell locomotion. We examined two such structures, lamellipodium and adhesion plaques, and found that lamellipodia area was coordinately reduced with MT dynamics. No effect was detected on adhesion plaque density or distribution. In time-lapse recordings, MTs did not penetrate into the lamellipodium of untreated cells, suggesting that MTs affect lamellipodia either through their interaction with factors at the base of the lamellipodium or by releasing factors that diffuse into the lamellipodia. In support of the latter hypothesis, when all MTs were rapidly depolymerized by 20 microM nocodazole, we detected the rapid formation of exaggerated protrusions from the leading edge of the cell. Our results show for the first time a linear relationship between MT dynamics and the formation of the lamellipodium and support the idea that MT dynamics may contribute to cell locomotion by regulating the size of the lamellipodium, perhaps through diffusable factors.     

Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid

We have examined the ryanodine receptor, Ca(2+)-ATPase, calsequestrin and phospholamban mRNA levels in the left ventricles of pacing-induced heart failure and norepinephrine infusion dogs. The heart failure dogs showed a decrease in the levels of ryanodine receptor and Ca(2+)-ATPase mRNAs. Norepinephrine infusion caused a reduction of Ca(2+)-ATPase mRNA but no change in ryanodine receptor mRNA. There was a corresponding reduction of the immunoreactive Ca(2+)-ATPase protein levels in both heart failure and norepinephrine infusion animals compared to controls. In contrast, the mRNAs of calsequestrin and phospholamban were unchanged in dogs with either congestive heart failure or norepinephrine infusion. Thus, since norepinephrine infusion and congestive heart failure produced similar reductions of Ca(2+)-ATPase mRNA and protein, we postulate that the down-regulation of Ca(2+)-ATPase in congestive heart failure may be caused, at least in part, by sympathetic stimulation that occurs in heart failure.     

Influence of purified apoprotein E on platelet activation induced by serotonin

In what ways can a premenopausal woman be pregnant after a breast carcinoma's treatment? A survey organized in 1985 by the French Gynecologic Association gave us the opinion of 316 gynecologists: 50% of them disagreed with this pregnancy because of an accrued risk of relapse. 68 observations were collected including 41 of full term pregnancies; the survival curves were, in each group, similar to those of the controls at the same stage. The interruption doesn't improve the prognosis. This confirms the whole of the published series: a subsequent pregnancy doesn't seem to affect the prognosis of a breast carcinoma; it is important to take the contraception into account so that this pregnancy shall be really desired.     

Antitumor responses induced by transgenic expression of CD40 ligand

Because CD40 ligand (CD40L) is a co-stimulator molecule for multiple components of the immune response, we wanted to determine whether transgenic expression of the molecule would increase immune responses against a weakly immunogenic murine tumor, neuro-2a. Tumor cells were transduced with a retroviral construct containing the CD40L gene and co-injected with variable numbers of non-CD40L transduced cells into syngeneic mice. Mice injected with cells that expressed CD40L had a significant reduction in average tumor size as compared to controls (p < 0.0001). In addition, survival of the neuro-2a/CD40L mice was 48 days versus 34 days for the neuro-2a/neo controls (p < 0.02). Expression of CD40L by less than 1.5% of neuro-2a cells was sufficient for significant antitumor effects (p < 0.001). These antitumor effects protected mice from subsequent challenge with parental neuro-2a cells. The protective effects of CD40L were associated with systemic immunomodulation. In vivo depletion of CD8+ cells abrogated the CD40L-mediated antitumor effects. Analysis of spleens from CD40L-protected animals showed increased numbers of CD4+ and CD8+ cells, the majority of which co-expressed the activation marker CD25. In addition, an increased number of antigen-presenting cells (APCs) expressed the co-stimulatory molecule CD86. These experiments illustrate that transducing even a small percentage of tumor cells with CD40 ligand can create a long-lasting systemic immune response capable of impeding growth of unmodified neuroblastoma cells.     

A trimeric, alpha-helical, coiled coil peptide: association stoichiometry and interaction strength by analytical ultracentrifugation

To examine the effects of TNF-alpha on luteal functions, TNF-alpha was injected into the ovary of rabbits on the 7th day of pseudopregnancy (Day 7). The animals were laparotomized under general anesthesia, and 1 x 10(4) IU TNF-alpha dissolved in PBS was injected into the ovary. On Days 8 and 10, the blood progesterone (P) concentration was determined. The mean blood P level was 10.31 ng before the administration of TNF-alpha on Day 7. On Day 8, the mean blood P level was 11.22 ng in the control group, while it was markedly reduced to 1.29 ng in the TNF-alpha administration group. On Day 10, the mean blood P level was 6.80 ng in the control group and 5.49 ng in the TNF-alpha group. These results suggest that the capacity for P secretion of the corpus luteum, which reached a degenerative stage by TNF-alpha administration, can be recovered.     

Rpb3, stoichiometry and sequence determinants of the assembly into yeast RNA polymerase II in vivo

Stoichiometry of the third largest subunit (Rpb3) of the yeast RNA polymerase II is a subject of continuing controversy. In this work we utilized immunoaffinity and nickel-chelate chromatographic techniques to isolate the RNA polymerase II species assembled in vivo in the presence of the His6-tagged and untagged Rpb3. The distribution pattern of tagged and untagged subunits among the RNA polymerase II molecules is consistent with a stoichiometry of 1 Rpb3 polypeptide per molecule of RNA polymerase. Deletion of either alpha-homology region (amino acids 29-55 or 226-267) from the Rpb3 sequence abolished its ability to assemble into RNA polymerase II in vivo.     

Death of solid tumor cells induced by Fas ligand expressing primary myoblasts

Anticancer therapy for solid tumors suffers from inadequate methods for the localized administration of cytotoxic agents. Fas ligand (FasL) has been reported to be cytotoxic to a variety of cells, including certain tumor cell lines. We therefore postulated that myoblasts could serve as non-transformed gene therapy vehicles for the continuous localized delivery of cytotoxic anticancer agents such as FasL. However, contrary to previous reports, fluorescence activated cell sorting (FACS) analyses revealed that both primary mouse and human myoblasts express Fas, the receptor for FasL. To avoid self-destruction and test the cytotoxic potential of myoblasts, the cells were isolated from mice deficient in Fas (lpr/lpr), the mouse counterpart of human autoimmune lymphoproliferative syndrome (ALPS). These primary mouse myoblasts were transduced with a retroviral vector encoding mouse FasL and expression of a biologically active and soluble form of the molecule was confirmed by the apoptotic demise of cocultured Fas-expressing Jurkat cells, the standard in the field. To test whether the lpr myoblasts expressing FasL could be used in anticancer therapy, human rhabdomyosarcoma derived cell lines were assayed for Fas and then tested in the apoptosis coculture assay. The majority of Fas-expressing muscle tumor cells were rapidly killed. Moreover, FasL expressing myoblasts were remarkably potent; indeed well characterized cytotoxic antibodies to Fas were only 20% as efficient at killing rhabdomyosarcoma cells as FasL expressing myoblasts. These findings together with previous findings suggest that primary myoblasts, defective in Fas but genetically engineered to express FasL, could function as potent anticancer agents for use in the localized destruction of solid tumors in vivo by three synergistic mechanisms: (1) directly via Fas/FasL mediated apoptosis, (2) indirectly via neutrophil infiltration and immunodestruction, and (3) as allogeneic inducers of a bystander effect via B and T cells.     

Learned helplessness in the rat: Time course, immunization, and reversibility.

Previous research has shown that rats, like dogs, fail to escape following exposure to inescapable shock. 3 experiments were conducted with a total of 121 male Sprague-Dawley rats to further explore parallels between rat and dog helplessness. The failure to escape did not dissipate in time; Ss failed to escape 5 min, 1 hr, 4 hrs, 24 hrs, and 1 wk after receiving inescapable shock. Ss that first learned to jump up to escape were not retarded later at barpressing to escape following inescapable shock. Failure to escape could be broken up by forcibly exposing the S to an escape contingency. Therefore, the effects of inescapable shock in the rat parallel learned helplessness effects in the dog. (23 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Strain differences in histopathologic, hematologic, and blood chemistry changes induced in mice by a technical and a purified preparation of 2,4,5-trichlorphenoxyacetic acid

Including controls, 978 mice were studied. On days corresponding to days 6 through 14 of pregnancy, groups of pregnant and nonpregnant CD-1 mice and male and nonpregnant female dihybrid cross F2 mice received by gavage 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) ranging in dosage from 30 to 140 mg/kg. Some groups received a technical preparation containing 97.9 +/- 0.4% 2,4,5-T and some a purified preparation containing 99 +/- 0.3% 2,4,5-T. Mice were sacrificed when they became moribund and at 1, 2, 4, 6, 8, and 11 days after beginning treatment. Sick or moribund mice sacrificed after 2-9 doses of 2,4,5-T often showed severe myocardial lesions, hypocellularlity of the bone marrow, and depletion of lymphocytes in the thymus, spleen or lymph nodes. They also showed marked hematologic and blood chemistry changes. Treated mice remaining healthy showed few or no lesions or blood chemistry changes, but often developed a mild anemia attributable to a hemolytic effect of 2,4,5-T. The incidence of animals becoming moribund was less than 1% in the CD-1 mice, including those given 140 mg/kg, and 53-82% in groups of male and female F2 mice receiving 120 mg/kg 2,4,5-T. The incidence of moribund mice tended to be higher in male than in female F2 mice and in those given the purified compound. These findings indicate that impairment of maternal health by severe lesions early in gestation is not the primary cause of an increase in incidence of fetal abnormalities observed in mice given 2,4,5-t. they also indicate that the lesions are due primarily to 2,4,5-T, rather than contaminants in the technical preparation, and illustrate the importance of using more than one strain of mouse in a toxicologic or teratologic study.     

Assembly of focal adhesions: progress, paradigms, and portents

Receptor-mediated assembly of an adhesion plaque occurs through an ordered series of steps, and intermediate assemblies can be identified. The recent demonstration of some of these partial reactions in permeabilized cells predicts that cell-free reconstitution of adhesion plaque assembly is an attainable goal. Newly discovered cryptic actin-binding sites in vinculin and ezrin, two proteins recruited to adhesion sites, suggest that actin-binding proteins are targets for the signals generated by adhesion receptors.     

Protoporphyrin IX sensitized photohemolysis: stoichiometry of the reaction and repair by reduced glutathione

Protoporphyrin IX acts as a sensitizer in the photohemolysis of bovine erythrocytes by binding to a limited number of membrane sites. The cholesterol-specific antibiotic lucensomycin competes with protoporphyrin in binding to the membranes. The possibility of cholesterol peroxidation as a primary event in photohemolysis is supported by the repairing effect of exogenous cholesterol and by the increased susceptibility of the photosensitized erythrocytes to lucensomycin. Glutathione, if present within the erythrocyte, postpones the onset of lysis; if added after irradiation, it may repair the membrane damage and prevent hemolysis. This effect appears to be related to a redox reaction (possibly involving glutathione peroxidase) between reduced glutathione and the cholesterol peroxide molecules.     

Effects of conditioned fear on responsiveness to pain: Long-term retention and reversibility by naloxone.

The effect of a fear CS on responsiveness to pain was examined in 3 experiments with 146 Long-Evans hooded rats. In Exp I, a CS that signaled shock attenuated freezing in response to shock, with the attenuation occurring several minutes after the shock. Naloxone blocked the effect of the CS. The effect of the CS, including its reversibility by naloxone, was retained over a 90-day interval. Exp II showed that this effect on freezing was due to associative fear conditioning rather than blocking of conditioning to context by a novel cue. In Exp III, presenting a fear CS just prior to administering a tailflick (radiant heat) test of nociception increased the tailflick latencies (i.e., the fear CS apparently induced hyperalgesia rather than analgesia). Because this result makes it difficult to interpret the change in freezing observed in Exp I as reflecting antinociception, it raises questions about how pain might differentially affect different measures of pain responsiveness. A memory hypothesis is advanced to resolve the different effects obtained with the freezing and tailflick tests. (29 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Caspase 1-independent IL-1beta release and inflammation induced by the apoptosis inducer Fas ligand

Anchietia salutaris tea is traditionally used in Brazil to treat allergies, suggesting it contains compounds with antagonistic activity on the allergic mediators. We have evaluated extracts and semi-purified fractions of Anchietia salutaris as a source of compounds having this type of antagonism on the contraction induced in guinea-pig lung parenchymal strips and on platelet aggregation and shape change. After 10 min pre-incubation dichloromethane extracts containing 30 or 100 microg mL(-1) inhibited the contraction induced by prostaglandin D2 (PGD2) in guinea-pig lung parenchymal strips with dose ratios (DR) of 0.76+/-0.14 and 0.93+/-0.19, respectively; the amount of inhibition depended both on the concentration and on the time of pre-incubation (DR after 30 min pre-incubation was 1.21+/-0.51). The dichloromethane extract and its semi-purified fractions also inhibited the contractions induced by U46619, a more potent, stable, synthetic agonist of thromboxane A2 (TxA2) prostanoid (TP) receptors, the receptors acted upon by PGD2 to produce lung contractions. The dichloromethane extract did not inhibit the lung parenchymal contractions induced by histamine, leukotriene D4 (LTD4) or platelet-activating factor (PAF). Platelet aggregation induced by U46619, adenosine 5'-diphosphate (ADP) or PAF was not inhibited by the dichloromethane extract. Indeed, the extract potentiated platelet aggregation induced by low concentrations of these agonists and also potentiated the shape change induced by U46619. These results imply that the dichloromethane extract of Anchietia salutaris and its semi-purified fractions contain an active principle that competitively inhibits TxA2 TP receptors, the stimulation of which causes lung parenchymal contraction. The inhibition seems to be selective for this receptor subtype, because the extract fails to inhibit platelet aggregation or shape change. This provides additional support of earlier reports suggesting the occurrence of TP receptor subtypes.     

Endogenous opiate reward induced by an enkephalinase inhibitor, thiorphan, injected into the ventral midbrain.

Opiates are known to be reinforcing when injected into the ventral tegmental area (VTA). The present study, with 87 female Sprague-Dawley rats, produced conditioned reinforcement with local injections of exogenous d-ala–2-met–5-enkephalinamide (DALA), a potent analog of met-enkephalin, and with thiorphan, an enkephalinase inhibitor that protects endogenous opiates from enzymic degradation. In a conditioned place perference paradigm, Ss received injections of DALA (1.0, 3.0, or 8.0 μg), thiorphan (60 μg), and/or naloxone (10 μg), or saline vehicle. Conditioned reinforcement was obtained with 8.0 μg of DALA and also with thiorphan but not with thiorphan plus naloxone. This suggests that reward can be generated by endogenous opiates in the VTA. Tests during the light phase and dark phase suggested that diurnal periodicity may play a role in opiate reward. It is concluded that the VTA can generate conditioned reward through transmitter–receptor interaction involving an endogenous opiate substrate that is probably enkephalinergic. (29 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)