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PubMed2004

Genotoxicity of goniothalamin in CHO cell line.

Goniothalamin (GTN) is a styrylpyrrone derivative from Goniothalamus umbrosus and other Annonaceae species. It has been shown to have anti-cancer and apoptosis-inducing properties against various human tumour and animal cell lines. The compound has also been shown to be active in vivo against DMBA-induced rat mammary tumours and was reported as an anti-fertility agent in rats. The aim of our study was to assess the genotoxicity of GTN in CHO cells using the UKEMS guidelines. A metabolic activation fraction (S9) was prepared according to standard methods. The methylthiazoletetrazolium (MTT) screening assay was then carried out to determine the cytotoxicity index (IC50) of GTN. The average IC50 value was 12.45 (+/- 3.63)microM. The mitotic index (MI) assay was then performed to determine the clastogenicity indices (MI(C25), MI(C50) and MI(C100)) of GTN. The chromosome aberration (CA) induction assay using air-dried metaphase spread was then performed to investigate the clastogenic effects of goniothalamin. Benzo[a]pyrene (BaP) and ethylmethanesulphonate (EMS) were used as positive controls in the presence and absence of S9 metabolic activation, respectively. The anti-genotoxicity effect of GTN was also assessed using a combination of GTN and EMS, and GTN and BaP. Dose-responses of CA frequencies were determined for both, the genotoxicity and anti-genotoxicity effects. GTN on its own and when combined with positive controls, was found to induce and enhance CA, respectively. Chromatid and whole chromosome breaks/gaps, as well as interchanges, endoreduplications and ring chromosomes were the main types of aberration induced by GTN. The overall clastogenic effect of GTN was statistically significant. In conclusion, GTN is potentially a genotoxic or clastogenic substance without any anti-genotoxic properties.

Animals↗
PubMed2006

Assessment of genotoxicity of 14 chemical agents used in dental practice: ability to induce chromosome aberrations in Syrian hamster embryo cells.

To assess the genotoxicity of 14 chemical agents used as locally applied agents in dental practice, the ability of these agents to elicit chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Chromosome aberrations in SHE cells were induced by treatment with three of eight chemical agents used as endodontic medicaments, i.e. ethylenediaminetetraacetic acid (EDTA), formocresol (a mixture of formalin and tricresol), and sodium arsenite. The other five chemical agents, i.e. chloramphenicol, p-chlorophenol, p-phenolsulfonic acid, sodium hypochlorite, and tetracycline hydrochloride exhibited a negative response for chromosome aberrations. Assessment of three dyes used for disclosing dental plaque showed chromosome aberrations induced by basic fuchsin but not by acid fuchsin and erythrosine B. Three local anesthetics, lidocaine hydrochloride, prilocaine hydrochloride, and procaine hydrochloride, were negative for chromosome aberrations. Among the ten chemical agents that exhibited a negative response in the assay, p-chlorophenol, sodium hypochlorite, and erythrosine B induced chromosome aberrations in SHE cells when treated in the presence of exogenous metabolic activation. The percentages of cells with polyploidy or endoreduplication were enhanced by formocresol, sodium arsenite, p-chlorophenol, p-phenolsulfonic acid, sodium hypochlorite, erythrosine B, prilocaine hydrochloride, and procaine hydrochloride in the absence or presence of exogenous metabolic activation. Our results indicate that the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

Anesthetics↗
PubMed2006

Progeny of germ line knockouts of ASI2, a gene encoding a putative signal transduction receptor in Tetrahymena thermophila, fail to make the transition from sexual reproduction to vegetative growth.

The ciliated protozoan Tetrahymena has two nuclei: a germ line micronucleus and a somatic macronucleus. The transcriptionally active macronucleus has about 50 copies of each chromosome. At sexual reproduction (conjugation), the parental macronucleus is degraded and new macronucleus develops from a mitotic product of the zygotic micronucleus. Development of the macronucleus involves massive genome remodeling, including deletion of about 6000 specific internal eliminated sequences (IES) and multiple rounds of DNA replication. A gene encoding a putative signal transduction receptor, ASI2, (anlagen stage induced 2) is up-regulated during development of the new macronuclei (anlagen). Macronuclear ASI2 is nonessential for vegetative growth. Homozygous ASI2 germ line knockout cells with wild type parental macronuclei proceed through mating but arrest at late macronuclear anlagen development and die before the first post-conjugation fission. IES elimination occurs in these cells. Two rounds of postzygotic DNA replication occur normally in progeny of ASI2 germ line knockouts, but endoreduplication of the macronuclear genome is arrested. The germ line ASI2 null phenotype is rescued in a mating of a knockout strain with wild type cells.

Animals↗
PubMed2006

Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF.

SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5'-TATCGATA) and DRE-like (5'-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter-luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter-lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF.

Animals↗
PubMed2000

Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay.

The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.

Aneuploidy↗
PubMed1998

The underlying structure of the subtelomeric region detected by microphotometrical scanning and chromosome graphic image analysis.

Previous research showed that the microphotometrical scanning of T-banded subtelomeric regions reveals the presence of specific patterns of the Giemsa stain density distributions as detected in chromosomes of normal human lymphocytes and CHO cells. Analyses with this method of the T-banded subtelomeric segments of CHO endoreduplicated chromosomes confirmed that these density patterns replicate in a similar way in sister chromosomes. Besides, the specific removal of portions of the subtelomeric segments appearing as tiny holes located where these density patterns are found suggested that both phenomena are related. The possible connection of these findings to the molecular structure of the subtelomeric region is briefly discussed.

Animals↗
PubMed1996

Cytogenetic effects of piperonyl butoxide and safrole in CHO-K1 cells.

Recently, hepatocarcinogenicity in rats and mice was reported with regard to the methylenedioxyphenyl compound, piperonyl butoxide (PB), which is used as a synergist for pyrethrins and related insecticides. Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs) due to PB were investigated using CHO-K1 cells with or without rat liver S9 fraction (S9); at the same time, the effects of safrole (SF), a methylenedioxyphenyl compound and a weak hepatocarcinogen, were also examined. PB (0.25 and 0.3 mM) and SF (0.8 mM) caused a slight but significant increase in SCEs followed by a cell-cycle delay in the 3-h treatment without S9. In the presence of S9 (4.5%), the cytotoxicity of PB or SF was weakened greatly or slightly, the top dose capable of cell division was raised to 0.6 mM (2-fold) or 1 mM, respectively. PB with S9 induced SCE at doses of 0.4 and 0.5 mM, and caused endoreduplications (ERDs, 7%) at a dose of 0.6 mM, while SF caused a dose-related significant increase in SCE at all doses used (0.4-1 mM) with S9. Genotoxicity of the metabolites of PB or SF was cleared by changing the dose of S9 (1.5-9%) while holding the dose of each chemical constant. In the case of SF (0.6 mM), induction of SCE, ERD and cell-cycle delay intensified almost in a dose-effect relationship, and CAs and a high level of ERD (14%) were caused by a 9% dose of S9. The concentration of unchanged SF in the incubated medium was certainly in inverse proportion to the dose of S9. This strongly suggests that the metabolites of SF are genotoxic. In the case of PB (0.3 mM), no positive responses were produced in the cultures, even with a high level of S9, though the amount of unchanged PB left in the incubated medium was very slight. This indicates that the metabolites of PB may not be genotoxic. In conclusion, PB and SF are possible to somewhat induce SCE at high dose(s) in the absence of S9, and the genotoxic effects of SF are more intensified in the presence of S9 than in its absence, while PB is probably no genotoxic in the presence of sufficient metabolic activation.

Animals↗
PubMed1997

Comparative evaluation of the in vitro micronucleus test and the in vitro chromosome aberration test: industrial experience.

Because of its rapidness, simplicity and potential for automation, the measurement of micronucleated cells in vivo is not only equivalent to the analysis of chromosome aberrations, but often even preferred within routine genotoxicity testing. In order to evaluate the correlation between the in vitro micronucleus assay (MNT) and the in vitro chromosome aberration test (CA), we collected data from four pharmaceutical companies obtained either in Chinese hamster cell lines (CHO-K5, CHO-K1, V79) or in human peripheral blood lymphocytes. Among the 57 compounds included in this comparison, 45 compounds gave rise to concordant results in both assays (26 compounds negative in both assays; 19 compounds positive in both assays). The high percentage of concordance, i.e. about 79% is very promising and can be even increased to about 88% by omitting the 3 aneugenic compounds and 2 compounds inducing endoreduplicated chromosomes which were found positive only in the in vitro MNT. The results are remarkable in particular considering that most of the compounds evaluated are 'standard' pharmaceutical compounds and thus are at most weak inducers of chromosome damage. Our comparison strongly supports that the in vitro micronucleus test is a suitable alternative to the in vitro chromosome aberration assay. Moreover, the MNT has the potential of not only detecting clastogens but additionally aneuploidy inducing chemicals.

Animals↗
PubMed2002

The origin of transformed cells. studies of spontaneous and induced cell transformation in cell cultures from marsupials, a snail, and human amniocytes.

Transformation of cells in culture is a model system for carcinogenesis, and two major theories (i.e., mutagenesis and aneuploidy) have emerged from in vitro and in vivo studies. A third view is presented here on the initial steps in the change of primary cells to extended life cells, and their change to immortalized cells. Both changes involve identical, microscopically visible cell abnormalities hitherto dismissed as cell degenerative characteristics. The major cell changes (i.e., giant cells, nuclear fragmentation to form multinucleated cells [MNC]) translated into genetic terms begin with the creation of polyploidy by DNA endoreduplication, followed by amitotic division of these giant cells to produce MNC. Individual nuclei, surrounded by a cell membrane, bud from the surface of the MNC, and represent the origin of the transformed cells. Induced budding by a protease treatment of MNC suggests that the extracellular matrix is an inhibitor of the budding process from human MNC. The production of the MNC is a genetic process determined by two abnormal events (i.e., overproduction of DNA and amitotic chromosomal segregation) during which there are possibilities for different genetic mechanisms to produce inherited variability within and between MNC. These concepts are discussed in regard to carcinogenesis, and by extension its possible prevention by use of the special cytopathic cell changes in carcinogen testing and in clinical screening programs.

Animals↗
PubMed2003

Hypocellular acute promyelocytic leukemia with a tetraploid clone characterized by two t(15;17).

We describe a case of hypocellular acute promyelocytic leukemia with a tetraploid clone characterized by two t(15;17). The large leukemia cells had a bizarre nuclear configuration and multiple Auer rods. A bone marrow biopsy specimen revealed a markedly hypocellular marrow (<10% cellularity) in the absence of myelofibrosis. Myelodysplastic features were not detected. Chromosome analysis of marrow cells revealed a karyotype of 92,XXYY,del(2)(q?),t(15;17)(q22;q21)x2. Interphase fluorescence in situ hybridization revealed that the marrow cells were composed of a tetraploid clone carrying double t(15;17) and normal diploid cells. The leukemia responded well to all-trans retinoic acid. We think that the tetraploidy could be caused by endoreduplication or endomitosis of the diploid clone with single t(15;17). The unique karyotype largely contributed to the cell morphology and marrow hypoplasia, while it may not have affected on the prognosis of the acute promyelocytic leukemia.

Bone Marrow Cells↗
PubMed2002

Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): association with cell senescence and tumour-promoting activities of stromal fibroblasts.

p21(Waf1/Cip1/Sdi1) is best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, but p21 also interacts with many other regulators of transcription or signal transduction. p21 induction, which is mediated by p53 and by p53-independent mechanisms, is essential for the onset of cell cycle arrest in damage response and cell senescence. The effects of p21 knockout in mice and its expression patterns in human cancer are consistent with a role for p21 as both a tumour suppressor and an oncogene. Several functions of p21 are likely to promote carcinogenesis and tumour progression. These include endoreduplication and abnormal mitosis that develop in tumour cells after release from p21-induced growth arrest, the ability of p21 to inhibit apoptosis through several different mechanisms, and its ability to stimulate transcription of secreted factors with mitogenic and anti-apoptotic activities. The latter effects of p21 show close resemblance to paracrine activities of senescent cells and to tumour-promoting functions of stromal fibroblasts. Therapeutic strategies targeting the oncogenic consequences of p21 expression may provide a new approach to chemoprevention and treatment of cancer.

Animals↗
PubMed1996

Potential role of DNA polymerase beta in gene therapy against cancer: a case for colorectal cancer.

Genetic instability characterized by the accumulation of mutations of tumor suppressor genes and oncogenes appears to be associated with carcinogenesis in colorectal and other cancers. Mutations of DNA polymerase beta (pol beta) and related chromosomal alterations appear to be consistent with the causal role of a "mutator phenotype' in carcinogenesis. However, homozygous knockout pol beta mutations appear to interfere with embryogenesis. Increased pol beta activity (i.e. relative to pol alpha activity) has been associated with cell cycle arrest. The related aphidicolin-resistant DNA replication has been observed primarily in differentiating cells, including the mammalian blastocyst, adrenal cortex, thyroid, anterior pituitary, and the mechanism of endoreduplication (amitotic over-replication of DNA) can be traced to lower eukaryotes. This increased activity in relation to terminal commitment is inconsistent with a simple "DNA repair' view of pol beta. It is therefore proposed that pol beta may play a more fundamental role in cellular differentiation through involvement in a putative subgenomic DNA replication-based model of terminal gene expression. Thus genetic instability, loss of differentiation, and carcinogenesis may result from aberration(s) or "derailment' of such replication-based mechanism of terminal gene expression. It is suggested to examine the relationship of DNA pol beta to genomic instability and carcinogenesis using genetic analyses and antisense technology with possible applications for gene therapy against colorectal cancer.

Animals↗
PubMed2002

The DNA replication-related element-binding factor (DREF) is a transcriptional regulator of the Drosophila myb gene.

Drosophila melanogaster possesses a single gene, Dm myb, that is closely related to the vertebrate proto-oncogene c-Myb, and its other family members (A-Myb and B-Myb), all of which encode transcription factors. Dm myb is expressed in all proliferating cells throughout development, and previous studies demonstrate that Dm myb promotes both S-phase and M-phase in proliferating cells, while preserving diploidy by suppressing endoreduplication. We have initiated a characterization of the mechanisms that regulate Dm myb expression, and we report here that the transcriptional activator DREF (the DNA replication-related element binding factor) activates Dm myb transcription via two binding sites located in the 5' flanking region; that the Dm myb promoter lacks a prototypical TATA box sequence and instead appears to use an initiator/downstream promoter element (Inr/DPE) type promoter; and that Dm myb expression is regulated at the translational as well as transcriptional level.

5' Flanking Region↗
PubMed2006

Evidence of different ploidy eggs produced by diploid F2 hybrids of Carassius auratus (female) x Cyprinus carpio (male).

Based on the presence of three types of eggs with different diameters 0.13, 0.17 and 0.2 cm, we made two crosses: F2 (female) x diploid red crucian carp (male), and F2 (female) x F10 tetraploid (male). The ploidy levels of the progeny of the two crosses were examined by chromosome counting and DNA content measurement by flow cytometer. In the offspring of the former cross, tetraploids, triploids, and diploid were obtained. In the progeny of the latter cross, tetraploids and triploids were observed. The production of the different ploidy level fish in the progeny of the two crosses provided a further evidence that F2 might generate triploid, diploid and haploid eggs. The presence of the male tetraploid found in F2 (female) x diploid red crucian carp (male) suggested that the genotype of XXXY probably existed in the tetraploid progeny. The gonadal structures of the tetraploids and triploids indicated that both female and male tetraploids were fertile and the triploids were sterile. We concluded that the formations of different ploidy level eggs from F2 were contributed by endoreduplication and fusion of germ cells.

Animals↗
PubMed2000

Mental retardation with rare fragile site expressed at 2q11.

Several rare autosomal folate sensitive fragile sites were reported in individuals with mental retardation, neurological abnormalities, and multiple congenital malformations. Only three of them: fra(11)(q22.3), fra(X)(q27.3) and fra(X)(q28), are known to be associated with mental retardation and phenotypic abnormalities. A possible association of the other rare fragile sites with idiopathic mental retardation is still being discussed. Here, a girl who has a fragile site at 2q11 with minor congenital anomalies and mental retardation is presented. This case has recalled the question of idiopathic mental retardation that might be the clinical expression of rare FSFS. Fragility was observed at 2q11 with a frequency of 3% in her cells along with a partial endoreduplication at 2 q11-->qter.

Child↗
PubMed2003

MYB and CBP: physiological relevance of a biochemical interaction.

Drosophila melanogaster possesses a single gene, Dm myb, that is closely related to the vertebrate family of Myb genes, which encode transcription factors involved in regulatory decisions affecting cell proliferation, differentiation and apoptosis. In proliferating cells, the Dm myb gene product, DMyb, promotes both S-phase and M-phase, and acts to preserve diploidy by suppressing endoreduplication. The CBP and p300 proteins are transcriptional co-activators that interact with a multitude of transcription factors, including Myb. In transient transfection assays, transcriptional activation by DMyb is enhanced by co-expression of the Drosophila CBP protein, dCBP. Genetic interaction analysis reveals that these genes work together to promote mitosis, thereby demonstrating the physiological relevance of the biochemical interaction between the Myb and CBP proteins within a developing organism.

Animals↗
PubMed1998

Therapeutic cytokine stimulation of thrombocytopoiesis.

Endogenous thrombopoietin (TPO) stimulates platelet production by inducing dose-dependent megakaryocyte development from early marrow hematopoietic progenitors and subsequent proliferation and endoreduplication. Recombinant human (rHu) TPO or pegylated recombinant human megakaryocyte growth and development factor (PEG-rHu MGDF) produce log-linear responses in experimental animals and humans when administered over the dose range of 0.05-25 micrograms/kg/day with respect to peak peripheral platelet counts (or peripheral platelet mass turnover) and marrow megakaryocyte volume, ploidy, number and mass. Other cytokines stimulating megakaryocytopoiesis in various species using different dosing regimens include Interleukin 1 (IL-1), Interleukin 3 (IL-3), Interleukin 6 (IL-6), Interleukin 11 (IL-11), stem cell factor (SCF), granulocytemacrophage colony stimulating factor, (GM-CSF), fusion proteins, such as promegapoietin, and a peptide agonist of TPO receptor. Whereas neither rHuTPO nor PEG-rHuMGDF induce platelet aggregation in vitro, they transiently enhance aggregatory responsiveness of platelets to physiologic agonists from several different experimental animals both in vitro and ex vivo. However, platelet recruitment into forming thrombus is not augmented by these agents when evaluated in quantitative rabbit or baboon models of platelet-dependent thrombus formation, except for the effect of platelet concentration per se. These findings indicate that appropriate dosing of these agents following marrow suppression prevents thrombocytopenia without increasing the risk of platelet-dependent thrombo-occlusive complications.

Blood Platelets↗
PubMed2002

Cyclin D-cdk4 is not a master regulator of cell multiplication in Drosophila embryos.

Inactivation of Cyclin E-Cdk2 is essential for a timely arrest of the epidermal cell proliferation program during Drosophila embryogenesis. E-type cyclin-cdk complexes are thought to be activated by D-types titrating away inhibitors and inducing cyclin E transcription by activating E2F transcription factors via Rb phosphorylation. Therefore, we have analyzed whether the developmentally controlled inactivation of Cyclin E-Cdk2 required for the epidermal cell proliferation arrest occurs as a consequence of Cyclin D-Cdk4 inactivation. However, preventing Cyclin D-Cdk4 inactivation by overexpression has a minimal effect on Cyclin E expression and does not interfere with the initial G1 arrest, while it readily induces the E2F target RnrS in arresting epidermal cells. Prolonged Cyclin D-Cdk4 overexpression eventually interferes with maintenance of quiescence in some cells. Moreover, in Cdk4 mutant embryos, some RnrS expression is still induced by Cyclin E overexpression, and endogenous Cyclin E expression as well as cell cycle progression is not affected, except for late aspects of the endoreduplication program. These findings argue against the proposed necessity of complete Rb inactivation by sequential phosphorylation by D- and E-type cyclin-cdk complexes. They demonstrate that Cyclin D-Cdk4 does not function as the master regulator of the embryonic cell proliferation program.

Animals↗