Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed toMycoplasma pneumoniae cells or membranes

Summary The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10⁵ to 10⁷ CFU of virulentMycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 μg per ml of purifiedM. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures. This investigation was supported in part by the National Institutes of Health (PHS Grant AI 12559), by a Biomedical Sciences Support Grant made to the University of Illinois School of Life Sciences, and by the University Research Board.     

Lack of squamous cell lung carcinoma in vitro chemosensitivity to various drug regimens in the adenosine triphosphate cell viability chemosensitivity assay.

A pilot study on squamous cell lung carcinoma (LC) chemosensitivity in adenosine triphosphate cell viability chemosensitivity assay (ATP-CVA) was performed. Besides the histological investigation, a modified ATP-CVA was used for the analysis of cancer cell chemosensitivity to four drug regimens, including topotecan, a promising agent for non-small-cell lung cancer (NSCLC) chemotherapy. Results of in vitro chemosensitivity testing showed chemoresistance or only weak response in the predominant amount of tumors.     

Linking tumor cell cytotoxicity to mechanism of drug action: an integrated analysis of gene expression, small-molecule screening and structural databases

An integrated, bioinformatic analysis of three databases comprising tumor-cell-based small molecule screening data, gene expression measurements, and PDB (Protein Data Bank) ligand-target structures has been developed for probing mechanism of drug action (MOA). Clustering analysis of GI50 profiles for the NCI's database of compounds screened across a panel of tumor cells (NCI60) was used to select a subset of unique cytotoxic responses for about 4000 small molecules. Drug-gene-PDB relationships for this test set were examined by correlative analysis of cytotoxic response and differential gene expression profiles within the NCI60 and structural comparisons with known ligand-target crystallographic complexes. A survey of molecular features within these compounds finds thirteen conserved Compound Classes, each class exhibiting chemical features important for interactions with a variety of biological targets. Protein targets for an additional twelve Compound Classes could be directly assigned using drug-protein interactions observed in the crystallographic database. Results from the analysis of constitutive gene expressions established a clear connection between chemo-resistance and overexpression of gene families associated with the extracellular matrix, cytoskeletal organization, and xenobiotic metabolism. Conversely, chemo-sensitivity implicated overexpression of gene families involved in homeostatic functions of nucleic acid repair, aryl hydrocarbon metabolism, heat shock response, proteasome degradation and apoptosis. Correlations between chemo-responsiveness and differential gene expressions identified chemotypes with nonselective (i.e., many) molecular targets from those likely to have selective (i.e., few) molecular targets. Applications of data mining strategies that jointly utilize tumor cell screening, genomic, and structural data are presented for hypotheses generation and identifying novel anticancer candidates.     

Determination of adenosine triphosphate in human semen to estimate the fertilizing potential and to quantify sperm antibodies

The measurement of the ATP content of fresh semen is as accurate as the estimation of sperm motility by conventional methods in discriminating between semen of fertile versus subfertile men. The ATP content of frozen thawed donor semen is correlated with the probability of conception per cycle of insemination. Exact quantification of cytotoxic sperm antibodies in serum is possible with the adenosine-triphosphate-release-cytotoxicity test, since measurement is free of the bias of microscopic examination. The procedure has been simplified by testing only one serum dilution and calculating the ‘sperm toxicity index’.     

Evolution of the adenosine triphosphate site and design of new inhibitors of DNA topoisomerases II

Summary DNA topoisomerase II is required for chromosome segregation. The enzyme expresses its biological activity upon ATP hydrolysis into ADP and inorganic phosphate. Both ATP and dATP are equivalent substrates, but the 8-azidoadenosine 5′-triphosphate is a poor substrate comparing to ATP for calf thymus DNA topoisomerase II. This result is interesting and can be used for the design of new inhibitors or new better substrates and furthermore for biocaptors.     

Evolution of the adenosine triphosphate site and design of new inhibitors of DNA topoisomerases II

DNA topoisomerase II is required for chromosome segregation. The enzyme expresses itsbiological activity upon ATP hydrolysis into ADPand inorganic phosphate. Both ATP and dATP areequivalent substrates, but the 8-azidoadenosine5-triphosphate is a poor substrate comparing toATP for calf thymus DNA topoisomerase II. Thisresult is interesting and can be used for thedesign of new inhibitors or new bettersubstrates and furthermore for biocaptors.     

Influence of adenosine cyclic nucleotide analogs on growth of human colon tumor cell lines

The influence of three analogs of cyclic adenosine monophosphate (cAMP) and theophylline on growth of colon tumor cell lines HT 29, LIM 1215 and COLO 206F was assessed by serial estimates of cell number. Administration of theophylline or analog of cAMP 8-bromo cAMP (8-br-cAMP) to actively replicating cultures resulted in a decrease in cell number of each cell line. In contrast analogs of cAMP, dibutyryl cAMP (db-cAMP) and chlorophenylthio cAMP (cp-cAMP) caused an increase in cell number of each cell line. This variation between analogs makes it difficult to draw conclusions regarding the influence of cAMP on cell growth when analogs are used to mimic the biological role of cAMP.     

Apoptosis in non-small cell lung cancer as related to drug resistance and prognosis

The percentage of apoptotic cells among the tumor cells (apoptotic index) was determined in a series of 178 non-small cell lung carcinomas with a long term clinical follow-up by a terminal desoxynucleotidyl transferase mediated dUTP nick end labelling technique. In survival analysis, we found no statistically significant correlation between the apoptotic index and survival times. We estimated also the sensitivity of specimens to doxorubicin by a short term test. Tumors with a high apoptotic activity were more sensitive to doxorubicin than tumors with a less apoptotic index. Thus, our data indicate that apoptosis may be involved in drug response of lung tumors and it could be useful for the chemotherapeutic strategy to design drugs which trigger apoptosis.     

The effect of phenformin and other adenosine triphosphate (ATP)-lowering agents on insulin binding to IM-9 human cultured lymphocytes

In the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM-9 human cultured lymphocytes. After a 24-hr preincubation, phenformin induced a twofold increase in specific 125I-insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between 125I-insulin binding and energy metabolism in IM-9 cells was investigated. The phenformin-induced increase in insulin binding to IM-9 cells was related to a time- and dose-dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2-deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM-9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a lowering of ATP levels.     

Combined therapy of p53-wt and drug in an orthotopic multidrug-resistant human lung cancer model

Balb/c nu/nu mice were inoculated intratracheally with multidrug-resistant human lung cancer cells GLK containing p53 mutation at codon 245 and treated with intratracheal instillation of p53-wt retroviral vector (pDOR53W) to increase cell chemosensitivity, and then with intraperitoneal injection of doxorubicin. 30 d after tumor cell inoculation, 75% of the control mice showed macroscopic tumors in the lung. Sole pDOR53W suppressed GLK tumor formation in 68 % of mice; sole doxorubicin 33. 3 % , but the combination of pDOR53W and doxorubicin 88.9%. The exogenous p53 sequence was detected and confirmed in the tumor that grew after treatment with pDOR53W retroviral vector by PCR and Southern blot hybridization with p53 cDNA. These results suggested that di-rect administration of a retroviral vector expressing p53-wt combined with treatment of anticancer agent was an effec-tive therapeutic method for multidrug-resistant human lung cancer.     

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 °C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.     

Ca2+ responses to acetylcholine and adenosine triphosphate in the otocyst of chick embryo

The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca²⁺-sensitive fluorescence measurements. Increases in [Ca²⁺]_i were evoked by the bath application of acetylcholine (1 μM or higher). The rise in [Ca²⁺]_i was due to the release of Ca²⁺ from intracellular Ca²⁺ stores, since the Ca²⁺ response occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolisehd the response to 10 μM acetylcholine; muscarine and carbamylcholine (100 μM each) evoked Ca²⁺ rises. Increases in [Ca²⁺]_i were also evoked by the bath application of ATP (10 μM or higher). The Ca²⁺ rise by ATP was evoked even in a Ca²⁺-free medium. Adenosine (500 μM) did not cause any Ca²⁺ response. Suramin and reactive blue 2 (200 μM each) completely blocked the Ca²⁺ response to 500μM ATP. Uridine triphosphate (500 μM) caused comparable Ca²⁺ responses with those to 500 μM ATP. These results suggested the involvement of P_2U purinoceptors. The potentiation of Ca²⁺ rise was observed when acetylcholine and ATP were co-applied at submaximal concentrations (10 μM and 100 μM, respectively). We conclude that undifferentiated cells in the otocyst epithelium have CaCa²⁺ mobilizing systems activated by acetylcholine and ATP. © 1995 John Wiley & Sons, Inc.     

Selection of tumor cell variants for resistance to tumor necrosis factor also induces a form of pleiotropic drug resistance

Summary This study has addressed the question of whether there may be some common mechanism underlying the induction or expression of acquired cytokine and drug resistance in a tumor cell line. This study employed the tumor-necrosis-factor(TNF)-sensitive U937 tumor cell line as a model system to determine if selection of a tumor cell variant for cytokine resistance would also result in drug resistance and vice versa. Variants were selected by culturing in the presence of purified recombinant TNF or a mixed-lymphokine-containing supernatant derived from concanavalin-A-stimulated peripheral blood lymphocytes. The resulting variants were resistant not only to TNF, but also to certain chemotherapeutic drugs. The variants were most resistant to colchicine and theVinca alkaloids, requiring drug concentrations 50- to 5000-fold higher to mediate levels of cytotoxicity comparable to that seen with the parental U937. The variants were moderately resistant to cycloheximide, actinomycin D, and mitomycin C. In contrast, these lines were relatively sensitive to doxorubicin or daunomycin. This phenomenon was not unique to U937 cells since we obtained a similar pattern of drug resistance by selecting TNF-resistant variants of the WEHI-164 tumor cell line. The cytokine-selected U937 variants were still lysed by NK cells, although they were somewhat less sensitive than the parental U937. Both variants were relatively resistant to lysis by activated macrophages, probably because of their TNF resistance. In an alternative selection procedure, U937 variants were derived by culturing in the presence of increasing concentrations of colchicine. The resulting variants were relatively resistant to TNF, providing further support for the existence of some common mechanism operating in induction or expression of acquired cytokine and drug resistance. The resistance mechanism apparently does not involve the P glycoprotein since the cytokine-selected U937 variants do not overexpress the mdr gene. This study has demonstrated that selection of TNF-resistant variants results in coexpression of a unique form of drug resistance that is characterized by resistance to microtubule-active drugs but not to the anthracycline antibiotics and is not associated with overexpression of the mdr gene.This work was supported by grant CA 47 669-01 awarded by the National Cancer Institute Nomenclature of variants: U9-LKR, U937 variant selected by lymphokines; U9-TR, U937 variant selected by tumor necrosis factor (TNF); WEHI-TR, WEHI-164 variant selected by TNF     

Inhibition of tumor cell growth by a human B-cell line

An Adenocarcinoma cell line (Breast-M) and an Epstein-Barr virus (EBV)-infected B-cell line (Hairy-BM) were established from breast tumor tissue. The Hairy-BM was CD20⁺, CD25 (Tac)+ and surface immunoglobulin (sIg)+. Hairy-BM suppressed the in vitro proliferation of Breast-M in a time and a dose-dependent manner. The suppression was also found in 5 different human tumor targets showing tumor-Hairy-BM binding, but not; in 2 murine tumor targets showing no significant tumor-Hairy-BM binding. Lytic activity of Hairy-BM was found only against Breast-M.Abbreviations sIg Surface immunoglobulin - CTL Cytotoxic T-cells - NK Natural killer - IL2 Interleukin 2 - LAK Lymphokine activated killer - CSN Culture supernatant - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoolium bromide - PCR Polymerase chain reaction - TIL Tumor-infiltrating lymphocytes - HCL Hairy cell leukemia - TNF Tumor necrosis factor     

Campylobacter jejuni adenosine triphosphate phosphoribosyltransferase is an active hexamer that is allosterically controlled by the twisting of a regulatory tail

Adenosine triphosphate phosphoribosyltransferase (ATP‐PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP‐PRT from the pathogenic ε‐proteobacteria Campylobacter jejuni (CjeATP‐PRT). This enzyme is a member of the long form (HisG_L) ATP‐PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP‐PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP‐PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP‐PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme.     

Midazolam induces cellular apoptosis in human cancer cells and inhibits tumor growth in xenograft mice

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosisinducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-X_L and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.     

Truncated midkine correlates with sensitivity to anticancer drugs and malignancy in human tumor cell line

Midkine (MK) is a heparin binding growth factor having functions of neurite-outgrowth, mitogenesis and tissue repair. This molecule is involved in tumor growth and metastasis. The MK molecule consists of five exons, but there is a truncated isoform, lacking exon 3. We established SW13 cells transfected with the human truncated MK cDNA. These cells were induced to undergo apoptosis by anticancer agents, cisplatin, etoposide (ETP), mitomycin C (MMC) and paclitaxel (PAX). Truncated midkine (tMK) suppressed cell death and helped the cells to be viable. When the cells were cultured on dishes coated with extracellular matrix molecules, spontaneous detachment occurred in the tMK expressing cells. Also tMK enhanced cell invasion. These results suggest that expression of tMK has cell-protective functions and plays important roles in carcinogenesis and malignancy. Furthermore, it is suggested that tMK has a greater ability of malignant transformation than full-length MK. Whether tMK is expressed or not will be useful information for improving cancer chemotherapy.