The induction of aneuploidy in cultured Chinese hamster cells by propionaldehyde and chloral hydrate

The induction of aneuploidy in cultured Chinese hamster cells by propionaldehyde (PA) and chloral hydrate (CH) has been studied. Chinese hamster embryonic diploid (CHED) cells were grown as a monolayer in cover glasses. Treatments were performed with doses of 5 x 10(-4), 1 x 10(-3) and 2 x 10(-3)% of PA for 3 h and doses of 1 x 10(-3), 2 x 10(-3) and 3 x 10(-3)% of CH for 1.5 h. Treatments with 2 x 10(-3)% of acetaldehyde (AA) for the same PA and CH treatments were used as positive controls. Untreated cultures were used as negative controls. PA induced chromosomal aberrations with the three doses employed although in a lesser degree than the positive control. CH induced chromosomal damage only with the two higher doses. No correlation was found between the amount of chromosomal damage induced and the doses of PA or CH employed. Both compounds increased the frequency of aneuploid cells in relation to untreated controls but not in relation to the positive control. However, neither PA nor CH significantly increased the frequencies of polyploid cells. These results indicate that aldehydes and chlorine-replaced aldehydes are strong inducers of aneuploidy despite some differences between PA or CH and AA regarding cytotoxicity and polyploidy induction.     

The induction of aneuploidy by 3-nitrobenzo[a]pyrene in Chinese hamster ovary cells

Nitrobenzo[a]pyrenes are found in urban air particulates and particulates from diesel exhaust, gasoline engines and wood burning stoves. Following exposure of Chinese hamster ovary cells (CHO-K1-BH4) to 3-nitrobenzo[a]pyrene (3-NB[a]P), cells with multiple nuclei and/or nuclei with multiple lobes were observed. When CHO cells were treated with 5 micrograms/ml 3-NB[a]P for 5 or 20 h, aneuploidy was noted in these cells at 24-96 h post-exposure. The addition of N6, O2'-dibutyryl adenosine 3':5'-cyclic monophosphate to 3-NB[a]P-exposed CHO cell cultures appeared to reduce the amount of aneuploidy in treated cultures. Structure--activity studies showed that 1-NB[a]P was a much less effective inducer of aneuploidy than 3-NB[a]P and 6-NB[a]P was ineffective. 1-, 3- and 6-nitrosobenzo[a]pyrenes were not effective inducers of aneuploidy in CHO cells, and aneuploidy was not observed in cultures treated with 3-NB[a]P in the presence of S9 activation. It appears that the parent 3-NB[a]P is responsible for producing aneuploidy in CHO cells.     

Chromosome aberration induction in Chinese hamster ovary cells by restriction enzymes with different methylation sensitivity

The isoschizomer pair MspI and HpaII were used to investigate whether the putative specificity of restriction endonucleases would be maintained when they were introduced into mammalian cells. Although both enzymes recognize the sequence CCGG, HpaII will cut only if the internal cytosine is unmethylated, whereas MspI will cut regardless of the methylation status. Cleavage results in a cohesive-end DNA double-strand break, which can lead to the formation of chromosome aberrations. Since mammalian DNA is heavily methylated, one would expect MspI to be much more effective than HpaII at inducing chromosome aberrations in Chinese hamster ovary cells. In fact, during G₁, MspI induced a >90-fold higher number of aberrations than did HpaII. Cell cycle studies indicated that during early S there was a 30-fold increase in HpaII-induced aberrations. This increase may be due to increased accessibility of replicating hypomethylated DNA. Cells that were treated with the demethylating agent 5-aza-2-deoxycytidine (AzdC) displayed only a moderate increase in HpaII-induced aberrations during G₁. This observation, together with the results of restriction enzyme analysis of genomic DNA, indicated that demethylation was incomplete. The effects of AzdC on the induction of aberrations by MspI suggested that AzdC increases chromatin accessibility. Our results were consistent with the expected specificity of MspI and HpaII. Thus, it appears that restriction endonucleases can play a useful role in determining the biological consequences of DNA double-strand breaks.     

Malsegregation as a possible mechanism of aneuploidy induction by metal salts in MRC-5 human cells

Many aneugenic compounds are known to affect one or more components of the mitotic apparatus leading to an erroneous migration of chromosomes. Malsegregation occurs when a chromosome (or a chromatid) fails to migrate and remains at the metaphase plate. Nondisjunction implies the lack of dissociation between sister chromatids and the migration of both together to the same pole. The aim of the present study was to provide evidence that the aneugenic effect of some metal salts is the consequence of malsegregation at anaphase and that it is not caused by nondisjunction mechanisms. The frequencies of lagging chromosomes at anaphase-telophase of mitosis, hypoploid metaphases, and kinetochore-positive micronuclei induced by cadmium chloride, potassium dichromate, and cacodilic acid (dimethylarsinic acid) in MRC-5 human cells were compared. The data indicate that all the tested compounds are able to induce aneuploidy in MRC-5 human cells. Positive, statistically significant correlations were found when kinetochore-positive micronuclei, hypoploidy, and lagging chromosome frequencies were compared. The results suggest that malsegregation is the main mechanism involved in the induction of aneuploidy by metal salts in MRC-5 cells.     

Inhibition of tumor induction by methylnitrosourea in resected urinary bladder]

In the present experiments the dependence of tumour induction upon the different phases of the cell cycle in the proliferating urinary bladder was examined. For stimulation of urothelial proliferation, a one-third resection of the bladder was performed in female Wistar rats. To synchronize the proliferating urothelial cells, hydroxyurea was given. The direct-acting urothelial carcinogen N-methyl-N-nitrosourea (MNU) was administered as a single intravesical dose during different cell cycle phases. The incidence of urothelial bladder tumors was 32.6% in the controls. By comparison, the tumour incidences were 18.9, 9.3, 21.7, 26.3, 25.0 and 30.0%, respectively, when MNU was instilled during the late Gi-, early and late S-, G2+M-, and early and late postmitotic phase. The results obtained from a total of 283 rats clearly document a cell cycle specific inhibition of tumour development in the proliferating urinary bladder particularly when the carcinogen was administered during the early S-phase. MNU has also been shown to produce mesenchymal tumours in the bladder (overall incidence: 4.9%) as well as urothelial tumours in the renal pelvis (3.2%) and ureters (1.4%).     

Early induction of genetic instability and apoptosis by arsenic in cultured Chinese hamster cells

In order to assess at what time from the beginning of exposure inorganic arsenic can give rise to genetic instability and trigger apoptosis, V79-C13 Chinese hamster cells were treated with 10 microM sodium arsenite for 24 h. Under these conditions, cell survival was >70% and cells showed neither an increase in chromosome aberration frequency nor a delay in cell cycle progression. Investigations, which were carried out every 6 h during the treatment, revealed an early appearance of genetically unstable cells, namely micronucleated, multinucleated and mononucleated 'giant' cells, as well as apoptotic cells. Indirect immunostaining using anti-beta-tubulin antibody showed severe alterations in spindle morphology after only 6 h treatment, when cells with small spindles whose poles were inside the metaphase plate appeared, and after 12 h treatment, when cells in which spindle assembly had completely failed were observed. These cells, unable to complete mitosis, underwent apoptosis. In fact, cells which turned out to be positive in the TdT-FragEL test had condensed chromatin arranged in metaphase-like plates; their maximum frequency was reached after 24 h treatment. A cytogenetic study was conducted at the end of the period of exposure to arsenic and after post-treatment incubation in fresh medium for up to 5 days. It showed that the percentage of cells with 21 chromosomes (modal number of the cell line) decreased, making way for aneuploid cells. Arsenic, therefore, induced early genetic instability or apoptosis in dividing cells. However, while apoptosis tended to cease when arsenic was removed from the culture medium, the acquired instability remained and propagated within the cell population.     

Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance.

The increased use of antibacterial and antifungal agents in recent years has resulted in the development of resistance to these drugs. The significant clinical implication of resistance has led to heightened interest in the study of antimicrobial resistance from different angles. Areas addressed include mechanisms underlying this resistance, improved methods to detect resistance when it occurs, alternate options for the treatment of infections caused by resistant organisms, and strategies to prevent and control the emergence and spread of resistance. In this review, the mode of action of antifungals and their mechanisms of resistance are discussed. Additionally, an attempt is made to discuss the correlation between fungal and bacterial resistance. Antifungals can be grouped into three classes based on their site of action: azoles, which inhibit the synthesis of ergosterol (the main fungal sterol); polyenes, which interact with fungal membrane sterols physicochemically; and 5-fluorocytosine, which inhibits macromolecular synthesis. Many different types of mechanisms contribute to the development of resistance to antifungals. These mechanisms include alteration in drug target, alteration in sterol biosynthesis, reduction in the intercellular concentration of target enzyme, and overexpression of the antifungal drug target. Although the comparison between the mechanisms of resistance to antifungals and antibacterials is necessarily limited by several factors defined in the review, a correlation between the two exists. For example, modification of enzymes which serve as targets for antimicrobial action and the involvement of membrane pumps in the extrusion of drugs are well characterized in both the eukaryotic and prokaryotic cells.     

Replication of minute virus of mice in Chinese hamster ovary fibroblasts

Minute virus of mice (MVM), a parvovirus, replicates in Chinese hamster ovary (CHO) cells. Although replication of the virus cannot readily be detected by haemagglutination, it can be measured by plaque assay on mouse strain LM cells.     

Enhancement of the clastogenicity of N-nitrosodialkylamines plus near-ultraviolet irradiation by ethanol in Chinese hamster lung fibroblasts

Early work from our laboratory showed a synergistic action of N-nitrosodialkylamines and near-ultraviolet light (UVA, 320–400 nm) to cause mutations in bacteria and phages. Recently we reported that N-nitrosodialkylamines + UVA induces chromosome aberrations in Chinese hamster lung cells in culture. We have now found that ethanol can potentiate this clastogenic action of N-nitrosodialkylamines + UVA. When the cells were treated with N-nitrosopyrrolidine (NPYR) or N-nitrosodiethylamine (NDEA) + UVA for 2 hours in the presence of 1% ethanol, approximately 2-fold increase in the numbers of cells with aberrant chromosomes was observed, compared to those found without the ethanol. NPYR/NDEA only, ethanol only, or ethanol + UVA did not cause the aberrations. The enhance-ment was dependent on the concentration of ethanol. Treatment of cells with ethanol before the NPYR + UVA was ineffective. By contrast, treatment of cells with NPYR + UVA and then with ethanol was as effective as with the simultaneous treatment. Methanol showed synergistic effects similar to those of ethanol, but mannitol did not. Intracellular hydrogen peroxide was found to be increased twofold over that in the background by a treatment with ethanol + UVA. The alcohol-mediated enhancement of the clastogenic action of N-nitrosodialkylamines + UVA may be a consequence of an increase in intracellular oxidative stress, or simply due to increased membrane permeability. Environ. Mol. Mutagen. 29:296-302, 1997 © 1997 Wiley-Liss, Inc.     

抗HER-2抗体chA21对人乳腺癌SKBR3细胞凋亡的诱导作用及其机制

目的探讨抗HER-2工程抗体chA21在体外对高表达HER-2的人乳腺癌SKBR3细胞凋亡的诱导作用及其分子机制。方法采用透射电镜和原位末端标记技术(TUNEL)观察和检测chA21对SKBR3细胞凋亡的诱导,采用免疫细胞化学技术检测凋亡相关基因bc l-2、bax、Fas及caspase-3表达的改变。结果chA21作用72 h,可见SKBR3细胞凋亡,chA21高浓度组(5.4mg/L)凋亡指数显著高于低浓度组(0.2 mg/L)(P<0.01);chA21处理组SKBR3细胞的bax、Fas及caspase-3表达增加,而bc l-2表达及bc l-2/bax比值降低,上述改变在chA21高、低两个浓度组间有显著差异(P<0.01)。结论chA21在体外可诱导SK-BR3细胞凋亡,其分子机制与调节凋亡相关基因bax、bc l-2、Fas及caspase-3的表达有关。     

A review of the symposium on aneuploidy: Etiology and mechanisms

A symposium titled “Aneuploidy: Etiology and Mechanisms” was held in Washington, D.C., in March, 1985. The stimulus for convening it was the growing concern that environmental agents with the capacity to induce aneuploidy can have detrimental effects on human health. Major components of the symposium were devoted to an analysis of human aneuploidy, mechanisms by which aneuploidy originates, and tests for the detection of agents that induce aneuploidy. This review outlines the content of the symposium, its conclusions, and major uncertainties in the field.     

Effect of treatment medium on induction of aneuploidy by nocodazole in Saccharomyces cerevisiae

While studying ways to improve responsiveness of Saccharomyces cerevisiae strain D61.M to agents that induce aneuploidy, we noted that nocodazole, which strongly induces aneuploidy when yeast cells are treated in yeast extract-peptone-dextrose (YEPD) medium, had no effect when a synthetic complete (SC) medium was used. Further study revealed that the presence of peptone was necessary for induction. Other aneuploidy-inducing agents, including ethyl acetate, acetone, and methyl benzimidazole-2-yl-carbamate (MBC), were equally active in either medium. Benomyl, which degrades to MBC, was less active in SC than in YEPD medium.     

The induction of aneuploidy by alkylated purines: effects on early and late cell cycle events

It has been shown that alkylated bases induce aneuploidy inmammalian cells in culture. The mechanism of action is not clear,however, data with 6-dimethyl amino purine (6DMAP) suggest thatthis analogue might act by affecting the cytoskeleton and proteinkinases involved in cell cycle regulation (cdc2/p34). The aimof this work was to study the effect of O⁶methylguanine (O⁶meG),O⁶ethylguanine (O⁶etG) and 6DMAP on DNA synthesis induced bygrowth factors in two cell lines, 3T3 and CHEF/18 fibroblasts,which respond in opposite ways to substances affecting the cytoskeleton,colchicine and cholera toxin: DNA synthesis initiation is stimulatedin 3T3 cells and inhibited in CHEF/18 cells by such compounds.Our results indicate that O⁶meG and O⁶etG behave like choleratoxin, in as much as they inhibit DNA synthesis induced by epidermalgrowth factor plus insulin in CHEF/18 cells, and stimulate itin 3T3 cells. 6DMAP behaves differently and inhibits DNA synthesisin both cell lines. The inhibition (or stimulation) was greaterwhen alkylated bases were added before S phase started, suggestingthat these compounds might affect early events of the cell cycle.In CHEF/18 cells the three alkylated bases were able to induceaberrant metaphases and ana-telophases with different efficiency(70–100%). The effect was not dependent on the G₁–Sblock and it was reversible even after cell commitment to DNAsynthesis. ⁴To whom correspondence should be addressed     

Chemically induced aneuploidy in mammalian cells: mechanisms and biological significance in cancer

A growing body of evidence from human and animal cancer cytogenetics indicates that aneuploidy is an important chromosome change in carcinogenesis. Aneuploidy may be associated with a primary event of carcinogenesis in some cancers and a later change in other tumors. Evidence from in vitro cell transformation studies supports the idea that aneuploidy has a direct effect on the conversion of a normal cell to a preneoplastic or malignant cell. Induction of an aneuploid state in a preneoplastic or neoplastic cell could have any of the following four biological effects: a change in gene dosage, a change in gene balance, expression of a recessive mutation, or a change in genetic instability (which could secondarily lead to neoplasia). To understand the role of aneuploidy in carcinogenesis, cellular and molecular studies coupled with the cytogenetic studies will be required. There are a number of possible mechanisms by which chemicals might induce aneuploidy, including effects on microtubules, damage to essential elements for chromosome function (ie, centromeres, origins of replication, and telomeres), reduction in chromosome condensation or pairing, induction of chromosome interchanges, unresolved recombination structures, increased chromosome stickiness, damage to centrioles, impairment of chromosome alignment, ionic alterations during mitosis, damage to the nuclear membrane, and a physical disruption of chromosome segregation. Therefore, a number of different targets exist for chemically induced aneuploidy. Because the ability of certain chemicals to induce aneuploidy differs between mammalian cells and lower eukaryotic cells, it is important to study the mechanisms of aneuploidy induction in mammalian cells and to use mammalian cells in assays for potential aneuploidogens (chemicals that induce aneuploidy). Despite the wide use of mammalian cells for studying chemically induced mutagenesis and chromosome breakage, aneuploidy studies with mammalian cells are limited. The lack of a genetic assay with mammalian cells for aneuploidy is a serious limitation in these studies.     

Internalization and limited processing of basic fibroblast growth factor on Chinese hamster lung fibroblasts

Using either acidic (pH 2.5) or trypsic treatments, we demonstrated that 125I-labeled basic Fibroblast Growth Factor (125I-bFGF) was submitted to an internalization process on responsive Chinese hamster lung fibroblasts (CCL39) at 37 degrees C. Various experiments based on the measurement of cell-associated radioactivity, as well as on research of degradated products of 125I-bFGF in cellular supernatants, showed that most of the internalized radioactivity remained intracellularly located after up to 5 hr of incubation. Analyses of this radioactivity by NaDodSO4-PAGE revealed the presence of labeled peptides issued from the limited processing of the native 125I-bFGF form (17 kD) and whose molecular weights were estimated to be 9 and 6 kD. Kinetic experiments indicated that proteolysis of the 125I-bFGF began early on incubation (less than 30 min) and led to a prolonged preservation of the 9- and 6-kD peptides which were still detectable after 13 hr of incubation. Preincubation of the cells with different lysosomotropic agents completely inhibited the proteolysis, indicating that this event occurred probably in an intracellular acidic compartment. Two enzyme inhibitors, leupeptin and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), were also shown to interfere with the formation of both 9- and 6-kD peptides, thus suggesting a way to control the appearance of these fragments, and hence to determine their potential intracellular role.     

Enhancement of amyloid induction by amyloid fibril fragments in hamster

An extract (fibril amyloid-enhancing factor) prepared by sonication of a suspension of water-purified hamster AA-amyloid fibrils showed an amyloid-enhancing effect upon intraperitoneal injection into hamsters. The fibril amyloid-enhancing factor was identical to the original fibrils according to the results of infrared spectroscopy, gel filtration, gel electrophoresis, and Western blotting; although lyophilized samples of the extract did not show any green birefringence after staining with Congo red. From these results, it was concluded that fibril amyloid-enhancing factor represents small fragments of amyloid fibrils which enhance in vivo formation of amyloid fibrils.