Chinese Hamster Cells Research Articles

Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1mg/kg; intraperitoneal injection) at 18weeks of age, for 14days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased (P<0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P<0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P<0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P<0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity.

Repair of single-point mutations by polypurine reverse Hoogsteen hairpins.

Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.

Abstract 2418: Investigating p53 and other potential determinants of cell sensitivity to ATR inhibition by VE-821

Abstract Background: Defects in the multifactorial DNA damage response (DDR) are common in cancer, making them vulnerable to inhibitors of other components of the DDR. ATR is a key component of the DDR, signalling ssDNA, arising from stalled replication forks, resected double strand breaks (DSBs) and NER intermediates to activate cell cycle arrest at the S/G2 checkpoints and initiate homologous recombination repair (HRR). Tumors often have a dysfunctional G1 checkpoint e.g. due to p53 mutations or other DDR defects. ATR inhibition of the S/G2 checkpoints and HRR has the potential to exploit these defects and selectively target cancer. VE-821 is a potent ATR inhibitor under advanced pre-clinical investigation Hypotheses: 1) Cells with dysfunctional p53 will be more sensitive to ATR inhibition. 2) Defects in other DDR components will sensitise cells to ATR inhibition. Methods: The survival of isogenic p53 mutant/null and wt/corrected human cell lines (HCT116 p53+/+ and p53-/- or U2OS p53WT and p53DN (dominant negative)) and a panel of repair defective Chinese hamster ovary and lung fibroblast cells was determined using colony formation after 24 hours exposure VE-821 alone or in the presence of gemcitabine or ionising radiation. Results: There was no significant difference in sensitivity to VE-821 between p53 wt or mutant/null cells in either HCT116 (LC50 = 2.13 and 4.56 µM, respectively) or U2OS pairs (LC50 = 2.54 and 3.34 µM, respectively). However, 1 µM VE-821 significantly potentiated the antimetabolite, gemcitabine, in HCT116 p53-/- (4.3-fold) but not p53+/+ cells (1.5-fold), and enhanced cell kill by 100 nM gemcitabine 40-fold in U2OS p53DN cells but only 3-fold in U2OS p53WT cells. Chinese hamster ovary cells defective in XRCC1 (single stranded break repair) were more sensitive to single agent VE-821 than the parental wt cells (35% and 55% survival at 10 µM, respectively). Data on other repair-defective cell lines will be presented. Conclusions: p53 status is not a determinant of sensitivity to VE-821 as a single agent but VE-821 preferentially sensitises cells lacking functional p53 to gemcitabine. This supports the hypothesis that p53 status may be predictive of cellular response to ATR inhibition in combination with certain chemotherapies. Furthermore, data using Chinese hamster cell lines indicate that DDR defects may be exploited by ATR inhibition. Citation Format: Fiona K. Middleton, Tao Chen, John R. Pollard, Nicola J. Curtin. Investigating p53 and other potential determinants of cell sensitivity to ATR inhibition by VE-821. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2418. doi:10.1158/1538-7445.AM2014-2418

Failure to Replicate: A Sign of Scientific Misconduct?

Repeated failures to replicate reported experimental results could indicate scientific misconduct or simply result from unintended error. Experiments performed by one individual involving tritiated thymidine, published in two papers in Radiation Research, showed exponential killing of V79 Chinese hamster cells. Two other members of the same laboratory were unable to replicate the published results in 15 subsequent attempts to do so, finding, instead, at least 100-fold less killing and biphasic survival curves. These replication failures (which could have been anticipated based on earlier radiobiological literature) raise questions regarding the reliability of the two reports. Two unusual numerical patterns appear in the questioned individual’s data, but do not appear in control data sets from the two other laboratory members, even though the two key protocols followed by all three were identical or nearly so. This report emphasizes the importance of: (1) access to raw data that form the background of reports and grant applications; (2) knowledge of the literature in the field; and (3) the application of statistical methods to detect anomalous numerical behaviors in raw data. Furthermore, journals and granting agencies should require that authors report failures to reproduce their published results.

XRCC5 as a risk gene for alcohol dependence: evidence from a genome-wide gene-set-based analysis and follow-up studies in Drosophila and humans.

Genetic factors have as large role as environmental factors in the etiology of alcohol dependence (AD). Although genome-wide association studies (GWAS) enable systematic searches for loci not hitherto implicated in the etiology of AD, many true findings may be missed owing to correction for multiple testing. The aim of the present study was to circumvent this limitation by searching for biological system-level differences, and then following up these findings in humans and animals. Gene-set-based analysis of GWAS data from 1333 cases and 2168 controls identified 19 significantly associated gene-sets, of which 5 could be replicated in an independent sample. Clustered in these gene-sets were novel and previously identified susceptibility genes. The most frequently present gene, ie in 6 out of 19 gene-sets, was X-ray repair complementing defective repair in Chinese hamster cells 5 (XRCC5). Previous human and animal studies have implicated XRCC5 in alcohol sensitivity. This phenotype is inversely correlated with the development of AD, presumably as more alcohol is required to achieve the desired effects. In the present study, the functional role of XRCC5 in AD was further validated in animals and humans. Drosophila mutants with reduced function of Ku80-the homolog of mammalian XRCC5-due to RNAi silencing showed reduced sensitivity to ethanol. In humans with free access to intravenous ethanol self-administration in the laboratory, the maximum achieved blood alcohol concentration was influenced in an allele-dose-dependent manner by genetic variation in XRCC5. In conclusion, our convergent approach identified new candidates and generated independent evidence for the involvement of XRCC5 in alcohol dependence.

XRCC2 rs3218536 polymorphism decreases the sensitivity of colorectal cancer cells to poly(ADP-ribose) polymerase 1 inhibitor

Single nucleotide polymorphisms (SNPs) are associated with the development of certain types of cancer. The present study aimed to investigate the association between X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2) SNPs and colorectal cancer (CRC) cell sensitivity to the poly(ADP-ribose) polymerase (PARP) 1 inhibitor olaparib (AZD2281). SNaPshot® analysis of XRCC2 SNPs was performed in five CRC cell lines. The AZD2281-sensitivities of the CRC cells were also analyzed using MTT assays. The effect of AZD2281 on XRCC2 and PARP1 expression was investigated in the five cell lines using quantitative polymerase chain reaction and western blot analyses. Parallel investigations were performed using a cisplatin (DDP) model of DNA damage. The XRCC2 rs3218536 SNP was found to be associated with the LoVo microsatellite instability CRC cell line. The relative rate of growth inhibition was found to be lower in the LoVo cells following treatment with AZD2281 compared with the other four cell lines (P=0.002). Furthermore, the XRCC2 mRNA level in the LoVo cells was observed to be significantly higher than that in the other four cell lines (P<0.05). Similar results were found using the DDP model of DNA damage (P<0.05). The present study indicated that the XRCC2 rs3218536 polymorphism decreases the sensitivity of CRC cells to AZD2281.

A tamoxifen derivative, N,N-diethyl-2-[4-(phenylmethyl) phenoxy] ethanamine, selectively targets P-glycoprotein-positive multidrug resistant Chinese hamster cells

DPPE, a tamoxifen derivative with antihistamine activity, was previously shown to potentiate the toxicity of chemotherapeutic drugs. Recently, a Phase III clinical study using doxorubicin with DPPE demonstrated significant increase in the overall survival of breast cancer patients. In this study we examined the effects of DPPE alone on the growth of drug sensitive and P-gp positive CHO cell line. Our results demonstrate DPPE is selectively toxic to P-gp positive cells and the sensitivity to DPPE alone correlated with the levels of P-gp expression. Moreover, in MDR cells, DPPE-induced apoptosis was significantly reduced with Bcl2 overexpression and in the presence of P-gp ATPase inhibitor, PSC833. Furthermore, knockdown of P-gp expression in MDR cells with P-gp-siRNA reversed DPPE sensitivity and increased their sensitivity to doxorubicin and taxol but not to cisplatin. The addition of DPPE to membrane fractions led to dose-dependent increase in P-gp ATPase that was inhibited with PSC833. Moreover, incubation of P-gp positive cells with DPPE led to a significant increase in superoxide levels and a drop in cellular ATP and GSH pools that were reversible with inhibitors of P-gp ATPase. The combined presence of DPPE and the mitochondria electron transport complex III inhibitor, antimycin A, synergized in their effects on the growth of MDR cells but had no effect on the growth of parental drug sensitive cells. Collectively, the results of this study provide a possible mechanism that may be relevant to the clinical results of DPPE in breast cancer trial and demonstrates DPPE as P-gp collateral sensitivity drug.

Neonatal Genome-Wide Methylation Patterns in Relation to Birth Weight in the Norwegian Mother and Child Cohort

Although epigenetic regulation plays a critical role in embryonic development, few studies have examined the relationship of epigenome-wide methylation with fetal growth. Using the Infinium HumanMethylation450 BeadChip (Illumina, Inc., San Diego, California) in a substudy of 1,046 infants from the Norwegian Mother and Child Cohort Study (MoBa) enrolled between 1999 and 2008, we examined epigenome-wide cord blood DNA methylation in relation to birth weight. In multivariable-adjusted robust linear regression models, we identified differential methylation at 19 cytosine-guanine dinucleotides (CpGs) associated with either decreased (AT-rich interactive domain 5B (MRF1-like) (ARID5B), 2 CpGs) or increased (x-ray repair complementing defective repair in Chinese hamster cells 3 (XRCC3), 4 CpGs) birth weight. ARID5B knockout mice have less adipose tissue and significantly lower weight in the postnatal period. XRCC3 plays a key role in the maintenance of chromosome stability and the repair of DNA damage. Although there are fewer data on the other implicated genes, many of these genes have been shown to have roles in developmental processes. This constitutes the largest and most robust study of birth weight using an epigenome-wide methylation platform and offers potential insights into epigenetic mechanisms of fetal growth.

MiR-7 inhibits colorectal cancer cell proliferation and induces apoptosis by targeting XRCC2

BackgroundAnalysis using publicly available algorithms predicts that X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2), a key component in the homologous recombination repair pathway, is a potential target of micro-ribonucleic acid-7 (miR-7). Some studies have shown that both miR-7 and XRCC2 are associated with cancer development. For this purpose, we searched for the possible relationship between miR-7 and XRCC2 in the development of colorectal cancer (CRC).MethodsmiR-7 expression was assessed in CRC specimens and cell lines using real-time polymerase chain reaction (PCR). Luciferase reporter assay was used to confirm the target associations. The effect of miR-7 on cell proliferation and apoptosis was confirmed in vitro by the methylthiazol tetrazolium (MTT) assay, colony formation assay, and flow cytometry. Gene and protein expression were examined using real time PCR and western blotting, respectively.ResultsmiR-7 was downregulated in CRC specimens and cell lines, and targeted the 3′ untranslated region of XRCC2. miR-7 overexpression reduced cyclin D1 expression and increased p21, caspase-3, and BAX expression, which subsequently inhibited CRC cell proliferation and induced CRC cell apoptosis. However, XRCC2 can repress the inhibitory effects of miR-7 on proliferation.ConclusionOur findings suggest that miR-7 plays a protective role by inhibiting proliferation and increasing apoptosis of CRC cells. It may identify new targets for anti-cancer treatment.

Expression of base excision repair key factors and miR17 in familial and sporadic breast cancer

Understanding of BRCA1/2 interaction with the base excision repair (BER) pathway could improve therapy based on ‘synthetic lethality', whose effectiveness is based on homologous recombination deficiency in cells lacking functional BRCA genes. However, poly (ADP-ribose) polymerase (PARP) inhibitors failed in some patients and for this reason we explored BER key enzyme expression. In this study, the expression of BER enzymes (redox factor 1/apurinic-apyrimidinic endonuclease 1 (REF1/APEX1), NTH endonuclease III-like 1 (NTHL1), 8-oxoguanine DNA glycosylase (OGG1), PARP1) and of the scaffold protein XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1) were investigated in familial (BRCA-related and not) and sporadic breast cancer cases. Furthermore, miR17 expression was measured because of its role in the epigenetic regulation of BRCA1. Gene expression was evaluated in BRCA1-mutated cell lines, SUM149PT and SUM1315MO2, and in a BRCA1-proficient triple-negative MDA-MB-231 cell line. A cohort of 27 familial and 16 sporadic breast cancer patients was then examined to confirm results obtained from the cell line model. APEX1/REF1 was found to be upregulated in familial BRCA-wild-type and sporadic cases, indicating this enzyme as a potential therapeutic target. Furthermore, XRCC1 was overexpressed in BRCAX patients; consequently, we suggest to test the effectiveness of inhibitors targeting two different BER components in preclinical studies. XRCC1, which is also involved in the non-homologous end-joining pathway, was found to be downregulated in BRCA2-related patients concurrently with no change in PARP1 expression. Interestingly, no difference in PARP1 and miR17 expression was found in BRCA-related and sporadic breast cancer cases. PARP1 and miR17 could therefore be further investigated as molecular biomarkers of ‘BRCAness' phenotype, indicating patients which could really benefit from PARP inhibitor therapies.

Association study of c.910A&gt;G and c.1686C&gt;G polymorphisms in XRCC1 gene with risk of hepatocellular carcinoma in the Chinese population

XRCC1 (human X-ray repair complementing defective repair in Chinese hamster cell 1) gene is considered a potentially important gene influencing the risk of hepatocellular carcinoma (HCC). Our analyses detected two allelic variants of XRCC1, c.910A>G and c.1686C>G. We aimed to investigate whether these polymorphisms influence the risk of HCC. The association between the XRCC1 polymorphisms and the risk of HCC was analyzed in 719 patients and 662 controls by polymerase chain reaction-restriction fragment length polymorphism. Our data suggested that the genotypes and alleles of c.910A>G and c.1686C>G polymorphisms were statistically associated with the risk of HCC. For c.910A>G, the GG genotype was associated with increased risk of developing HCC compared with the AA wild genotype (OR = 1.95, 95%CI = 1.40-2.70, P < 0.0001). For c.1686C>G, the risk of HCC was significantly higher for the GG genotype compared with the CC wild genotype (OR = 1.89, 95%CI = 1.375-2.599, P < 0.0001). Significant differences in the risk of HCC were also found with other genetic models for these two SNPs. The G allele of both c.910A>G and c.1686C>G may contribute to the risk of HCC (G versus A: OR = 1.40, 95%CI = 1.20-1.64, P < 0.0001 and G versus C: OR = 1.38, 95%CI = 1.19-1.61, P < 0.0001, respectively). Our findings suggest that the c.910A>G and c.1686C>G polymorphisms of XRCC1 are associated with the risk of HCC in the Chinese population.

Role of Epidermal Growth Factor Receptor Signaling in the Interaction of Neisseria meningitidis with Endothelial Cells

Neisseria meningitidis, the causative agent of meningitis and septicemia, attaches to and invades various cell types. Both steps induce and/or require tyrosine phosphorylation of host cell proteins. Here, we used a phospho array platform to identify active receptor tyrosine kinases (RTKs) and key signaling nodes in N. meningitidis-infected brain endothelial cells to decipher RTK-dependent signaling pathways necessary for bacterial uptake. We detected several activated RTKs, including the ErbB family receptors epidermal growth factor receptor (EGFR), ErbB2, and ErbB4. We found that pharmacological inhibition and genetic ablation of ErbB receptor tyrosine phosphorylation and expression resulted in decreased bacterial uptake and heterologous expression of EGFR, ErbB2, or ErbB4 in Chinese ovary hamster (CHO-K1) cells, which do not express of EGFR and ErbB4; the decrease caused a significant increase in meningococcal invasion. Activation of EGFR and ErbB4 was mediated by transactivation via the common ligand HB-EGF (heparin-binding EGF-like ligand), which was significantly elevated in infected cell culture supernatants. We furthermore determined that N. meningitidis induced phosphorylation of EGFR at Tyr845 independent of ligand binding, which required c-Src activation and was involved in mediating uptake of N. meningitidis into eukaryotic cells. Increased uptake was repressed by expression of EGFR Y845F, which harbored a point mutation in the kinase domain. In addition, activation of ErbB4 at its autophosphorylation site, Tyr1284, and phosphorylation of ErbB2 Thr686 were observed. Altogether, our results provide evidence that EGFR, ErbB2, and ErbB4 are activated in response to N. meningitidis infection and shed new light on the role of ErbB signaling in meningococcal infection biology.

Natural and glucosyl flavonoids inhibit poly(ADP-ribose) polymerase activity and induce synthetic lethality in BRCA mutant cells

Poly(ADP-ribose) polymerase (PARP) inhibitors have been proven to represent superior clinical agents targeting DNA repair mechanisms in cancer therapy. We investigated PARP inhibitory effects of the natural and synthetic flavonoids (quercetin, rutin, monoglucosyl rutin and maltooligosyl rutin) and tested the synthetic lethality in BRCA2 mutated cells. In vitro ELISA assay suggested that the flavonoids have inhibitory effects on PARP activity, but glucosyl modifications reduced the inhibitory effect. Cytotoxicity tests of Chinese hamster cells defective in BRCA2 gene (V-C8) and its parental V79 cells showed BRCA2-dependent synthetic lethality when treated with the flavonoids. BRCA2 mutated cells were three times more sensitive to the flavonoids than the wild-type and gene complemented cells. Reduced toxicity was observed in a glucosyl modification-dependent manner. The present study provides support for the clinical use of new treatment drugs, and is the beginning of the potential application of flavonoids in cancer prevention and the periodic consumption of appropriate flavonoids to reduce cancer risk in individuals carrying a mutant allele of the BRCA2 gene.

Ruthenium(II)/4,6-dimethyl-2-mercaptopyrimidine complexes: Synthesis, characterization, X-ray structures and in vitro cytotoxicity activities on cancer cell lines

The complexes [RuCl(SpymMe2-N,S)2(NO)] (1) and [Ru(dppb)(SpymMe2-N,S)2] (2) (dppb=1,4-bis(diphenylphosphino)butane, SpymMe2=deprotonated 4,6-dimethyl-2-mercaptopyrimidine) were obtained and characterized by elemental analysis, spectroscopic techniques including X-ray crystallography, conductimetry and cyclic voltammetry. Energy Decomposition Analysis (EDA) calculation indicated strong intermolecular interaction between monomers of complex 1, as suggested by the 1H NMR spectrum of this complex, in chloroform solution. Additionally, preliminary tests were carried out, in order to evaluate the cytotoxicity activity on cancer cell lines of the new complexes, and the free ligands, against MDA-MB-231 (breast cancer), Hela (cervical cancer), U251 (glioma cancer) tumor cell lines and the V79 (Chinese lung fibroblast hamster) cell line. The best result was obtained for the complex 1 with IC50 value of 0.11±0.02, against MDA-MB-231 tumor cells.

Tumor-Targeting with Novel Non-Benzoyl 6-Substituted Straight Chain Pyrrolo[2,3-d]pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and Inhibition of de Novo Purine Nucleotide Biosynthesis

A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a-d with varying chain lengths (n = 5-8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a-d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a-d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a-d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a-d. Hydrolysis and subsequent coupling with diethyl l-glutamate and saponification afforded target compounds 3a-d. Compounds 3b-d showed selective cellular uptake via FRα and -β, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.

Synthesis and Anti‐Plasmodium Activity of Benzimidazole Analogues Structurally Related to Astemizole

A series of compounds structurally related to astemizole were designed and synthesized with the goal of determining their anti-Plasmodium activity. Several modifications of the astemizole structure, namely the removal of the 4-fluorobenzyl and/or 4-methoxyphenethyl moieties, substitution of the benzene ring of the benzimidazole scaffold, replacement of the fluorine atom in the 4-fluorobenzyl group, and variation of the 4-aminopiperidine moiety, were explored. In vitro evaluation of the anti-Plasmodium activity of these compounds using the ItG strain showed that astemizole and some of its structurally similar derivatives have IC50 values in the nanomolar range and exhibit toxicity towards the parasite over Chinese ovarian hamster (CHO) cells with a selectivity as high as 200. The presence of a secondary cyclic amine at position 2 and substitution with chlorine at positions 4 and 5 in the benzimidazole moiety are two modifications that resulted in potent and selective antimalarials based on astemizole.

Abnormal mitotic spindle assembly and cytokinesis induced by D-Limonene in cultured mammalian cells

D-Limonene is found widely in citrus and many other plant species; it is a major constituent of many essential oils and is used as a solvent for commercial purposes. With the discovery of its chemotherapeutic properties against cancer, it is important to investigate the biological effects of the exposure to D-Limonene and elucidate its, as yet unknown, mechanism of action. We reported here that D-Limonene is toxic in V79 Chinese hamster cells in a dose-dependent manner. Moreover, to determine the cellular target of D-Limonene, we performed morphological observations and immunocytochemical analysis and we showed that this drug has a direct effect on dividing cells preventing assembly of mitotic spindle microtubules. This affects both chromosome segregation and cytokinesis, resulting in aneuploidy that in turn can lead to cell death or genomic instability.

Genomes of Chinese hamster cell lines mapped

Genomes of Chinese hamster cell lines mapped

Mutagenicity tests for isepamicin (HAPA-B)

To detect mutagenic activity of isepamicin (HAPA-B), a new aminoglycoside antibiotic, we carried out several mutagenicity tests using microorganisms and cultured mammalian cells. Effects of the antibiotic on DNA-lesion induction and repair were examined using the Rec-assay with Bacillus subtilis and a sister-chromatid exchange test with cultured Chinese hamster cells. The drug caused no increase of mutagenicity indices in both tests, suggesting that the antibiotic has no DNA-damaging effect. To further investigate the effect of HAPA-B on gene-mutation, we performed the Ames Salmonella/microsome plate assay and the thioguanine resistance mutation test using cultured Chinese hamster cells. In both tests, the antibiotic induced no increase of mutation frequencies over spontaneous levels. Clastogenic activities of HAPA-B and its effect on chromosome disjunction were examined by the chromosomal aberration test using cultured Chinese hamster cells. After a short- or long-time exposure, the antibiotic induced neither structural chromosome aberrations nor an increase in the number of tetraploid cells. The negative results from these five test systems with different mechanisms strongly suggest the safety of HAPA-B regarding mutagenicity.

A paradoxical effect of hydrated C60-fullerene at an ultralow concentration on the viability and aging of cultured Chinese hamster cells

The effect of an aqueous solution of hydrated C60-fullerene (HyFn) on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during the slowing down of their proliferation within a single passage and the subsequent “aging” in the stationary phase of growth) of transformed B11-dii FAF28 Chinese hamster cells was studied. The final calculated concentration of HyFn in the growth medium was 10−19 M. A paradoxical result contrasting the available data on the absence of HyFn cytotoxicity at higher concentrations was obtained in our experiments: namely, HyFn decelerated cell proliferation (estimated by the growth of mass culture, as well as by the efficiency of colony formation) and accelerated the “stationary phase aging” of the cell culture. Moreover, repeated addition of an aqueous solution of HyFn (to the final calculated concentration of 10−19 M) to the cells that had already reached the stationary phase of growth caused a rapid (within no more than 24 h) death of a significant part of the cell population. The observed effect of HyFn at ultralow concentration is supposed to arise from the alterations in the properties of the water surrounding the fullerene molecule: namely, water becomes a donor and acceptor of electrons and regulates redox processes (especially those involving oxygen) in aqueous systems. This effect of HyFn at an ultralow concentration may be specific for transformed cells, and, therefore, experiments on normal fibroblasts with limited mitotic potential are planned as a continuation of the present study. It is also possible that the reported antiaging effect of HyFn in experimental animals is due to its anticancer, immunostimulatory, antiviral, and antibacterial properties manifested only at the whole-organism level.