Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.     

The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm-free, CheY- containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo-taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.     

Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

Inhalation toxicity of methylisocyanate: assessment of germ cell mutagenicity and reproductive effects in rats.

Adult male Wistar rats were exposed to methylisocyanate (MIC, 3.2 mg/l, single inhalation exposure for 8 min under static condition) or ethyl methanesulphonate (EMS, 150 mg/kg, single ip dose) for the assessment of germ cell mutagenicity and reproductive effects. Sequential matings of treated males with normal females on days 1-7, 8-14 and 15-21 post-exposure did not indicate any induction of dominant lethal mutation (increased frequency of preimplantation losses and early fetal deaths) by MIC but it was significantly induced by EMS as compared to respective controls. Males, necropsied after 21 days of exposure, showed no effect of MIC on epididymal sperm density and morphology. EMS also had no effect on sperm density but it significantly induced morphological abnormalities in sperm as compared to untreated controls. There was an acute and transitional reduction in reproductive performance (10-21%) of MIC-exposed males during days 1-14 post-exposure followed by recovery to the normal level during days 15-21 post-exposure. The progeny of MIC-exposed males was also normal in terms of litter size, litter weight, neonatal survival and body weight gain in litters up to 10 days post-partem. It is concluded with the evidence at hand that the observed failure of MIC to cause germ cell mutagenicity is related to its poor biodistribution to the target site(s) and a transient reduction in the reproductive performance of MIC-exposed males is a result of general stress and disconsolate copulation.     

Activation of mineralocorticoid agonist and antagonist specific receptors from rat kidney

The kinetics of saturation, as well as of denaturation, confirm the existence of two distinct mineralocorticoid receptor populations one each for the agonist aldosterone (MR2) and the antagonist RU 26752 (MR3) in rat kidney. Receptor activation in vitro was dependent upon the buffer, progressed just as well in the presence of the agonist and the antagonist, and was inhibited by molybdate. These necessitate a reassessment of both the importance of receptor activation in vitro and its possible contribution to hormone action in vivo.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Circulating concentrations of thyroid hormones in pregnant camels (Camelus dromedarius )

Blood samples from 16 female camels were collected at monthly intervals commencing from 60 d post. breeding until the last month of gestation. Two camels failed to conceive and two had unnoticed abortions. The average gestation period was 398+/-13 and 372+/-11 in camels bearing male and female fetus, respectively, with an overall mean of 383+/-9 d. Sera were analyzed for thyroxine (T(4)) and triiodothyronine (T(3)) by radioimmunoassay. Mean T(4) and T(3) concentrations varied from 76 to 116 ng/ml and 0.73 to 1.32 ng/ml, respectively, during various stages of gestation. In general, the T(4) and T(3) levels were higher during early pregnancy, with lowest values in the tenth month. T(4):T(3) ratio showed minor, nonsignificant fluctuations. Age of dam of sex of fetus had no effect on hormone levels. Similarly, hormone levels were not affected by failure of conception or by abortion.     

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.     

Binding properties of diethylcarbamazine

Diethylcarbamazine (DEC) reacted with liver cell plasma membrane of rodent hosts-cotton rat, albino rat and Mastomys natalensis exhibiting the presence of both saturable and unsaturable components. The presence of lectins or sugar derivatives did not affect the binding significantly. The drug showed similar binding pattern with serum but the saturation was reached at a much lower concentration of the ligand. Data obtained with a variety of macromolecules, particularly with the homopolymers of amino acids indicate that DEC does not require any specific constituent of the membrane for binding. The nonspecific nature of DEC binding does not provide any convincing clue for the accumulation of microfilariae specifically in the liver following the drug treatment.     

A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary. This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging Research to T. M. and HL35724 to B. W. EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells.