Utilization of Neural Stem Cell-Derived Models to Study Anesthesia-Related Toxicity and Preventative Approaches

Early-life stress has been shown in both preclinical and clinical studies to cause neuroanatomical and biological alterations and disruptions in homeostasis. These alterations can lead to dysfunction in critical regulatory systems and concomitant increases in risk for the development of pathology. The existing data from research using in vivo animal models have implicated some general anesthetics as being toxic to the developing brain and causing cognitive deficits later in life. Because of obvious limitations, it is not possible to thoroughly explore the effects of early-life stress—e.g., prolonged exposure to anesthetic agents—on neurons in vivo in human infants or children. However, the availability of stem cell-derived models, especially human embryonic neural stem cells, along with their capacity for proliferation and ability to differentiate, has provided a potentially invaluable tool for examining the developmental effects of anesthetic agents in vitro. This review focuses on how embryonic neural stem cells, when combined with biochemical, pathological, and pharmacokinetic assessments, might serve as a bridging platform to provide the most expeditious approaches toward decreasing the uncertainty in extrapolating preclinical data to the human condition. This review presents key concepts in stem cell biology with respect to the nervous system, presents an overview of neural development, and summarizes the involvement of neural cell types in developmental neurotoxicity associated with anesthetic exposure.     

Qualitative and quantitative procedures for health risk assessment.

Numerous reactive mutagenic electrophiles are present in the environment or are formed in the human body through metabolizing processes. Those electrophiles can directly react with DNA and are considered to be ultimate carcinogens. In the past decades more than 200 in vitro and in vivo genotoxic tests have been described to identify, monitor and characterize the exposure of humans to such agents. When the responses of such genotoxic tests are quantified by a weight-of-evidence analysis, it is found that the intrinsic potency of electrophiles being mutagens does not differ much for the majority of the agents studied. Considering the fact that under normal environmental circumstances human are exposed to low concentration of about a million electrophiles, the relation between exposure to such agents and adverse health effects (e.g., cancer) will become a 'Pandora's box'. For quantitative risk assessment it will be necessary not only to detect whether the agent is genotoxic, but also understand the mechanism of interaction of the agent with the DNA in target cells needs to be taken into account. Examples are given for a limited group of important environmental and carcinogenic agents for which such an approach is feasible. The groups identified are agents that form cross-links with DNA or are mono-alkylating agents that react with base-moieties in the DNA strands. Quantitative hazard ranking of the mutagenic potency of these groups of chemical can be performed and there is ample evidence that such a ranking corresponds with the individual carcinogenic potency of those agents in rodents. Still, in practice, with the exception of certain occupational or accidental exposure situations, these approaches have not be successful in preventing cancer death in the human population. However, this is not only due to the described 'Pandora's box' situation. At least three other factors are described. Firstly, in the industrial world the medical treatment of cancer in patients occurs with high levels of extremely mutagenic agents. Actually, both in number of persons and in exposure levels such medical treatment is the single largest exposure of humans to known carcinogens. Although such treatments are very effective in curing the tumor as present in the patient, the recurrence of cancer in those patients later in life is very high. In other words: "curing cancer is not the same as preventing cancer death in the human population". Secondly, the rate of cancer death in the human population is also determined by the efficacy in which other major causes of death are prevented. For instance, cardiovascular diseases are the major cause of death in humans in the industrialized world. There is evidence that the treatment of cardiovascular diseases is more successful than that of cancer. On a population level this will result in increase of cancer being the ultimate death cause. Finally, the improvement of medical treatment of diseases together with an improved quality of life will lead to increase average age of the population. Because the onset of most cancer is long after the exposure to carcinogens-in human often more than 30 years-cancer is predominantly a disease of the old age. This means that if the average age of human increases, there will be a selective preference of cancer becoming an even more important cause of death. This especially will be pronounced in those countries were the age distribution in a population is abnormal.     

A mutagenicity assessment of acetaldehyde

Acetaldehyde has been shown in studies by several different laboratories to be a clastogen (chromosome-breaking) and inducer of sister-chromatid exchanges in cultured mammalian cells (Chinese hamster cells and human lymphocytes). Although there have been very few studies in intact mammals, the available evidence suggests that acetaldehyde produces similar cytogenetic effects in vivo. The production of cytogenetic abnormalities may be related to the ability of acetaldehyde to form DNA-DNA and/or DNA-protein cross-links. Acetaldehyde apparently has not been evaluated for its ability to cause gene mutations in cultured mammalian cells, but it has been shown to produce sex-linked recessive lethals in Drosophila. In general, bacteria tests have been negative. Although acetaldehyde is a genotoxic cross-linking agent, it does not appear to cause DNA strand breaks. There were no studies available regarding the potential of acetaldehyde to produce genetic damage in mammalian germ cells in vivo. Most mutagenicity testing on acetaldehyde has been motivated by attempts to define the proximate mutagen in ethanol metabolism.     

Experimental work on induced mutations

The detection of changes in mutation rate in human populations remains extremely difficult. Thus estimation of genetic hazards of mutagens to man depends on extrapolation from experimental systems. Germ cells of animals show complex variations in sensitivity to mutagenic effects. Some agents predominantly affect stem cells or other immature germ cells, whereas others mainly affect later germ cell stages. Dose-response relations also vary both with the agent and with the stage or sex of germ cell treated. In man, in addition to single-gene defects and chromosome anomalies, conditions of complex or uncertain inheritance, such as congenital malformations, are clinically important. Genetic theory leaves unclear whether the incidence of these would be affected by a change in mutation rate. Recent research has shown that in mice the incidence of malformations is increased by exposure of the parents to mutagens, but the effect is small. Chromosomal non-disjunction is also clinically important. Again, recent research shows that its frequency can be changed by mutagens, but the effects vary with germ-cell stage. Thus, further research is needed to elucidate the relative contributions of different environmental mutagens to human genetic disease.     

The life span-prolonging effect of Sirtuin-1 is mediated by autophagy

The life span of various model organisms can be extended by caloric restriction as well as by autophagy-inducing pharmacological agents. Life span-prolonging effects have also been observed in yeast cells, nematodes and flies upon the overexpression of the deacetylase Sirtuin-1. Intrigued by these observations and by the established link between caloric restriction and Sirtuin-1 activation, we decided to investigate the putative implication of Sirtuin-1 in the response of human cancer cells and Caenorhabditis elegans to multiple triggers of autophagy. Our data indicate that the activation of Sirtuin-1 (by the pharmacological agent resveratrol and/or genetic means) per se ignites autophagy, and that Sirtuin-1 is required for the autophagic response to nutrient deprivation, in both human and nematode cells, but not for autophagy triggered by downstream signals such as the inhibition of mTOR or p53. Since the life span-extending effects of Sirtuin-1 activators are lost in autophagy-deficient C. elegans, our results suggest that caloric restriction and resveratrol extend longevity, at least in experimental settings, by activating autophagy.     

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods.     

The role of receptors in multistage carcinogenesis

The principal characteristic of neoplasia is its inherited alteration of genetic expression. The regulation of gene expression may be altered both by mutational events and by environmental mediators. During carcinogenesis the permanent alterations in genetic expression resulting from mutations occur primarily during the final stage of progression when biological malignancy becomes evident. During the preceding reversible stage of promotion, alteration and genetic expression are the result of the chronic stimulation of an altered (initiated) cell responding to the environmental mediator or promoting agent. A major mechanism of this effect occurs by receptors exhibiting specificity for the mediator and for their interaction with the genome. Withdrawal of the promoting agent prior to the genetic alterations characteristic of the stage of progression leads to a reversal of the effects of the promoting agent and the death by apoptosis of most cells in the stage of promotion. Carcinogenesis mediated by the chronic ligand (promoting agent)-receptor interaction increases the probability of the development of the stage of progression; thus alteration or prevention of the stage of promotion by removal of the promoting agent or inhibition of its action remains the best opportunity for cancer prevention. Application of the reversible promoting agent-receptor interaction to specific environmental circumstances where such plays a major role can lead to a more rational risk estimation of promoting agents for the human population.     

Contribution of human data to the analysis of human carcinogenesis

Human data strongly suggest that small doses or low concentrations of genotoxic agents cause only a relatively small number of human cancers. They emphasize the role of promotion, in particular that associated with cell proliferation. There is therefore a qualitative difference between high doses of genotoxic agents which provoke cell death and a compensatory increase in cell division, and low doses which do not. During the promotion phase, human data demonstrate the importance of induced genetic instability and defects in apoptosis as well as that of cell immortalization which play a main role for the accumulation in a cell genome of several specific lesions. Carcinogenesis is a complex process in which initial mutations do not appear to be a limiting or crucial step. This view is supported by the paramount influence of age on the induction by radiation of thyroid and breast cancer. It is also compatible with practical thresholds observed in subjects whose bones or liver were exposed to alpha-emitters, as well as with the curvilinearity in the leukemia incidence dose-response in the Japanese atomic bomb survivors. The linear no threshold model assumes that: 1) the probability of DNA lesion repair is constant whatever the dose and, hence, the number of lesions provoked in the same cell and the surrounding cells; 2) the probability for a damaged cell to evolve toward an invasive cancer is not influenced by the possible promotional effect of further irradiation or induced tissue proliferation, nor the control exerted by surrounding cells. These assumptions deserve a critical analysis.     

Genetic toxicology of ethylenediaminetetraacetic acid (EDTA)

EDTA and its salts have a number of applications in medicine and pharmacy. EDTA is used to remove calcium from the human body, and serves as an anticoagulant and as a detoxicant after poisoning by heavy metals. It is often used in analytical chemistry for complexometric titrations and many other purposes. Because the compound is of rather low toxicity, it is used as a food additive to bind metal ions. EDTA affects the inhibition of DNA synthesis in primary cultures of mammalian cells. This may be due to impairment of enzymes involved in DNA replication. Some early studies have shown that EDTA leads to morphological changes of chromatin and chromosome structure in plant and animal cells. These alterations consist of dispersion or swelling of chromosomes or a loss of interphase chromatin structure. For several test systems, a low chromosome-breaking activity of EDTA has been reported. A weak activity in the induction of gene mutations has also been observed. It is well established that EDTA influences chromosome breakage by mutagenic agents. In particular, when applied in combination with chemical mutagens, EDTA enhances mutagen-induced aberration frequencies. Furthermore, the chelating agent is able to increase the incidence of meiotic crossing-over. This has been demonstrated for many gene loci in Drosophila melanogaster, Chlamydomonas reinhardi, Neurospora crassa and Zea mays. EDTA interferes with DNA repair processes that take place after exposure to mutagens. In E. coli or Micrococcus radiodurans as well as in Chinese hamster cells, the fast repair component detectable after treatment with ionizing radiation or bleomycin is inhibited by EDTA. In plant cells exposed to gamma-rays, EDTA inhibits unscheduled DNA synthesis. The mechanism by which EDTA causes these effects remains poorly understood. The sequestering of metal ions by the chelating agent is obviously responsible for functional and structural alterations of the genetic material. Although EDTA produces a whole set of genetic effects it seems to be a harmless compound to man as far as genotoxicity is concerned. The data presently at hand, however, are not sufficient for a reliable risk assessment.     

Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms

Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. We studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. The inactive colchicine analogues, beta- and gamma-lumicolchicine, did not inhibit FeTf-induced TfR downregulation. Similarly, when cells were pretreated with taxol to stabilize microtubules, colchicine no longer inhibited FeTf-induced downregulation. Therefore, FeTf causes TfR downregulation in lymphoblastoid cells by a cytoskeleton-dependent mechanism. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. In other experiments, treatment of cells with both a phorbol diester and FeTf, either simultaneously or sequentially, produced additive effects on TfR expression. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.     

Cross-talk between cAMP and formylmet-leu-phe in human neutrophils: phosphorylation of a 52,000 molecular weight protein.

The mechanism of inhibition of neutrophil phagocytic functions by cAMP-elevating agents has not yet been clarified. In the present work, the effects of adenylate cyclase agonists on protein phosphorylation in the formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated human neutrophils were studied. Before stimulation, 32Pi-labelled cells were incubated with adenosine deaminase to remove the endogenously produced adenosine, an adenylate cyclase agonist itself. A protein of about 52,000 molecular weight was rapidly and transiently phosphorylated when neutrophils were stimulated with fMLP in the presence of isoproterenol, prostaglandin E1, histamine or 2-chloroadenosine. This phosphorylation was blocked by the antagonists of the receptors for the above-listed agents. No phosphorylation of the 52,000 molecular weight protein could be observed if either fMLP or the cAMP-elevating agent were applied alone. A calcium ionophore A23187 and dibutyryl-cAMP could replace fMLP and a cAMP-elevating agent, respectively. Phosphorylation of the 52,000 molecular weight protein was also demonstrated in cell lysates in the presence of cAMP, and in membrane preparations in the presence of the catalytic subunit of cAMP-dependent protein kinase. These data suggest that phosphorylation of the 52,000 molecular weight protein in intact cells is dependent on the cross-talk between the fMLP- and the cAMP-signalling pathways, and may thus be involved in the cAMP-regulatory mechanism.     

Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs

Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4(+) lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.     

Genetic toxicology of bleomycin.

Bleomycin (BLM), an antibiotic obtained from Streptomyces verticillus, is of significance as an antineoplastic agent. The compound is actually the mixture of some 200 related forms which differ from each other in the amine moiety. The drug, at low concentrations, can cause elimination of bases, particularly thymine. This causes strand breakage of DNA and inhibition of cell growth. The influence of BLM on cell growth may be unrelated to the effects on DNA. In general, mitotically dividing cells show more DNA damage than non-dividing cells. G2 seems to be the most sensitive phase indicating that cell death may not be related to a direct effect of BLM on DNA replication. The antibiotic shows specific effects on chromatin and causes chromosomal damage in all sub-phases of interphase. It can affect early prophase chromosomes also. Suggestion has been made that BLM-induced breakage and cell death are similar to those induced by densely ionizing radiations. Whereas the antibiotic affects the frequency of somatic crossing over and produces micronuclei, the data on mutation induction and production of sister-chromatid exchanges do not permit classifying BLM as a potent inducer of these phenomena. The genetic effects of BLM can be modified quantitatively by thiol compounds, caffeine, hyperthermia and H2O2. It is concluded that the available data do not permit assessment of genetic damage in the offsprings of BLM-treated patients. Such studies are urgently needed, as are the studies to find out the effects of BLM on meiotic phenomena.     

Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations

The alkylating agent busulfan (Myleran) adversely affects spermatogenesis in mammals. We treated male mice with single doses of busulfan in order to quantitate its cytotoxic action on spermatogonial cells for comparison with effects of other chemotherapeutic agents, to determine its long-term effects on fertility, and to assess its possible mutagenic action. Both stem cell and differentiating spermatogonia were killed and, at doses above 13 mg/kg, stem cell killing was more complete than that of differentiating spermatogonia. Azoospermia at 56 days after treatment, which is a result of stem cell killing, was achieved at doses of over 30 mg/kg; this dose is below the LD50 for animal survival, which was over 40 mg/kg. Busulfan is the only antineoplastic agent studied thus far that produces such extensive damage to stem, as opposed to differentiating, spermatogonia. The duration of sterility following busulfan treatment depended on the level of stem cell killing and varied according to quantitative predictions based on stem cell killing by other cytotoxic agents. The return of fertility after a sterile period did not occur unless testicular sperm count reached 15% of control levels. Dominant lethal mutations, measured for assessment of possible genetic damage, were not increased, suggesting that stem cells surviving treatment did not propagate a significant number of chromosomal aberrations. Sperm head abnormalities remained significantly increased at 44 weeks after busulfan treatment, however, the genetic implications of this observation are not clear. Thus, we conclude that single doses of busulfan can permanently sterilize mice at nonlethal doses and cause long-term morphological damage to sperm produced by surviving stem spermatogonia.     

Synergic action between tumor necrosis factor and endotoxins or poly(A·U) on cultured bovine endothelial cells

Summary In order to investigate whether direct effects on tumor vasculature may contribute to induction of necrosis of solid tumors in vivo, agents and combinations with an established different capacity to induce tumor necrosis were studied for their effects on endothelial cells in vitro. Tumor necrosis serum caused a marked inhibition of [³H]thymidine incorporation by bovine umbilical cord endothelial cells after 4h coincubation. Endotoxin was less inhibitory, whereas detoxified endotoxin and recombinant human tumor necrosis factor (rTNF) were hardly active in concentrations that can be achieved in vivo. Combinations of rTNF and (detoxified) endotoxin caused synergic inhibition. By 24h effects of the separate agents and synergic effects of the combinations were much stronger. The nontoxic dsRNA, poly(A·U), also had inhibitory activity, and acted synergistically with rTNF. Morphologically, a combination of endotoxin and rTNF but not the separate constituents induced marked cell detachment by 24 h, an indication of cell death. Whereas both endotoxin and rTNF inhibited DNA synthesis of human endothelial cells, the agents did not act synergistically on these cells. The ability of the agents and the combinations to affect endothelial cells in culture appeared to be well in line with their capacity to induce tumor necrosis. Data suggest that direct (synergic) effects on endothelium may contribute to the induction of vascular damage in tumors by (combinations of) the agents. The fact that endothelial cell death is only induced by the combinations and not by the separate agents in vivo, may be a cause of the greater therapeutic activity of the combinations in vivo. The synergy between rTNF and the other agents indicates that the agents act by different mechanisms.Supported by a grant of the Stichting Koningin Wilhelmina Fonds, Netherlands Cancer Foundation     

DNA damage and repair: consequences on dose-responses

Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and cancer. Single or double strand breaks, bases modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions formed in different proportions according to agents and to DNA sequence context. They can result in cell death or in mutational events which in turn may initiate malignant transformation. Normal cells are able to repair these lesions with fidelity or by introducing errors. Base excision (BER) and nucleotide excision (NER) repair are error-free processes acting on the simpler forms of DNA damage. A specialized form of BER involves the removal of mismatched DNA bases occurring as errors of DNA replication or from miscoding properties of damaged bases. Severe damage will be repaired according to several types of recombinational processes: homologous, illegitimate and site-specific recombination pathways. The loss of repair capacity as seen in a number of human genetic diseases and mutant cell lines leads to hypersensitivity to environmental agents. Repair-defective cells show qualitative (mutation spectrum) and quantitative alterations in dose-effect relationships. For such repair-deficient systems, direct measurements at low doses are possible and the extrapolation from large to low doses fits well with the linear or the linear-quadratic no-threshold models. Extensive debate still takes place as to the shape of the dose-response relationships in the region at which genetic effects are not directly detectable in repair-proficient normal cells. Comparison of repair mutants and wild-type organisms pragmatically suggests that, for many genotoxins and tissues, very low doses may have no effect at all in normal cells.     

Covalent binding of the benzamide RH-4032 to tubulin in suspension-cultured tobacco cells and its application in a cell-based competitive-binding assay

The benzamide, RH-4032, was found to be a potent antimicrotubule agent in tobacco (Nicotiana tabacum) cells. It strongly inhibited root growth and produced swollen club-shaped roots, an accumulation of cells in arrested metaphase, and loss of microtubules. RH-4032 inhibited the in vitro assembly of bovine tubulin into microtubules, with inhibition requiring a relatively long incubation period. Treatment of tobacco suspension-cultured cells or isolated bovine tubulin with [(14)C]RH-4032, and analysis of radiolabeled protein revealed a highly specific covalent attachment to beta-tubulin. Binding of [(3)H]RH-4032 in tobacco suspension-cultured cells was shown to be saturable and to be influenced by pre-incubation of the cells with various antimicrotubule agents: Binding of [(3)H]RH-4032 was inhibited by the benzamides, pronamide and zarilamide, the N-phenylcarbamate, chlorpropham, and the microtubule-stabilizing drug, paclitaxel, whereas trifluralin and amiprophosmethyl were not inhibitory. A common characteristic of agents that cause microtubule disassembly was a slight enhancement of [(3)H]RH-4032 binding at low concentrations, which did not occur with the microtubule-stabilizing agent paclitaxel. For structural analogs of RH-4032 and various N-phenylcarbamates, it was shown that the ability to inhibit binding of [(3)H]RH-4032 was correlated with the ability to inhibit tobacco root elongation. The results suggest a common binding site on beta-tubulin for RH-4032, pronamide, zarilamide, and chlorpropham, which is distinct from the binding site(s) for trifluralin and amiprophosmethyl. RH-4032 provides a unique approach to studying effects of antimicrotubule agents on plant cells by allowing competitive tubulin binding assays to be conducted in whole cells.     

Biochemical analysis of metastasis-related Ax actin in B16 mouse melanoma cells after chemical reversional modulation and of tumor progression-related A' actin in the ontogeny of human malignant melanoma

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A'.actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metastasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A' actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A' actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A' actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.     

Zoledronic Acid Produces Combinatory Anti-Tumor Effects with Cisplatin on Mesothelioma by Increasing p53 Expression Levels

We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.