Chinese Hamster Ovary CHO-K1 Cells Research Articles

Genotoxicity test of eight natural color additives in the Korean market.

BackgroundVarious natural color additives are preferred by many consumers over synthetic color additives because they are perceived to be safer. However, most do not have sufficient toxicity data for safety assurance. Color ingredients in particular have some structures suspected of being toxic. Eight natural color additives, gardenia red, blue, and yellow; lac color; cochineal extract; beet red; Curcuma longa Linne extract (Curcuma extract); and Monascus red, currently permitted for use in Korea, were selected and subjected to genotoxicity tests. Acceptable daily intake values have not been allocated to these color additives (except for cochineal extract) due to the lack of toxicity data. We used genotoxicity testing—the bacterial reverse mutation test (Ames test), in vitro mammalian chromosomal aberration test, and in vivo alkaline comet test—for minimum safety assurance.ResultsGardenia red and blue, cochineal extract, lac color, and beet red did not induce mutagenicity or chromosomal abnormalities. Gardenia yellow was mutagenic in the Ames test, but was not positive in the in vitro chromosomal aberration test or in vivo alkaline comet assay. Curcuma extract and Monascus red induced cytotoxicity in the Ames test at high concentrations in Salmonella typhimurium TA1537 and TA100, without showing mutagenicity. On cytotoxicity testing, Curcuma extract and Monascus red showed cytotoxicity at concentrations higher than 313 μg/ml in Chinese hamster ovary CHO-K1 cells and showed equivocal results in chromosomal aberration assay of the same cells. Curcuma extract and Monascus red produced significant increases in DNA damage at a dose of 2000 mg/kg b.w./day, and induced dose-dependent increases in % DNA in the tail and tail moment on in vivo comet assay.ConclusionsSix out of eight food colorants did not cause genotoxicity and cytotoxicity. However, Monascus red and Curcuma extract showed definite cytotoxicity and probable genotoxicity.

Self-Assembled Nanophotosensitizing Systems with Zinc(II) Phthalocyanine-Peptide Conjugates as Building Blocks for Targeted Chemo-Photodynamic Therapy

Two zinc(II) phthalocyanines (ZnPcs) substituted with a short peptide (Gly-Gly-Lys) with either a carboxyl or a carbamoyl group at the C-terminus and an appended biotin moiety were prepared and characterized. They could self-assemble into spherical nanoparticles, namely ZnPc-GGK(B)-COOH NP and ZnPc-GGK(B)-CONH2 NP, through noncovalent interactions, which encapsulated the hydrophobic ZnPc units in the core and exposed the biotin moieties on the surface. The zeta potential of ZnPc-GGK(B)-COOH NP in water was found to be -28 mV, whereas that of ZnPc-GGK(B)-CONH2 NP was in opposite sign (+15 mV), reflecting the different functionality at the C-terminus, which also greatly affected the stability of the self-assembled nanoparticles. The targeting effect of ZnPc-GGK(B)-COOH NP was examined against human hepatocellular carcinoma HepG2 cells, which overexpress biotin receptor, and Chinese Hamster Ovary CHO-K1 cells, which have a low expression of biotin receptor. This nanosystem was also coassembled with the chemotherapeutic doxorubicin (DOX) to form ZnPc-GGK(B)-COOH/DOX NP. Both ZnPc-GGK(B)-COOH NP and ZnPc-GGK(B)-COOH/DOX NP could induce photocytotoxicity and apoptosis on HepG2 cells with an IC50 value of 1.48 and 0.49 μM ZnPc, respectively. For the latter nanosystem, the ZnPc and DOX components induced cytotoxicity in a synergistic manner. The photodynamic and chemotherapeutic effects of these two nanosystems were also examined on nude mice bearing a human colorectal adenocarcinoma HT29 tumor. The ZnPc-GGK(B)-COOH/DOX NP exhibited a stronger tumor inhibition effect upon irradiation, demonstrating the presence of dual chemo-photodynamic therapeutic actions.

Different localization of lysosomal-associated membrane protein 1 (LAMP1) in mammalian cultured cell lines.

Lysosomal-associated membrane protein 1 (LAMP1) mainly localizes to lysosomes and late endosomes. We herein investigated the intracellular localization of lysosomal membrane proteins in five mammalian cultured cell lines. Rat LAMP1 fused to enhanced green fluorescent protein (EGFP) mostly accumulated at a particular cytoplasmic area and barely co-localized with LysoTracker® Red DND-99 in golden hamster kidney BHK-21 cells and Chinese hamster ovary CHO-K1 cells. Golden hamster, Chinese hamster, and human LAMP1-EGFP showed a similar intracellular distribution to rat LAMP1-EGFP in BHK-21 cells. Endogenous LAMP1 was also detected in a perinuclear area in BHK-21 cells and CHO-K1 cells, and co-localized with rat CD63-EGFP in BHK-21 cells. Moreover, rat LAMP1-DsRed-Monomer co-localized well with the human trans-Golgi network protein 2-EGFP in BHK-21 cells. These results reveal that LAMP1 predominantly localizes to the trans-Golgi network in BHK-21 cells.

Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica

A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.

Tumor protein D52 expression and Ca2+-dependent phosphorylation modulates lysosomal membrane protein trafficking to the plasma membrane

Tumor protein D52 (also known as CRHSP-28) is highly expressed in multiple cancers and tumor-derived cell lines; however, it is normally abundant in secretory epithelia throughout the digestive system, where it has been implicated in Ca(2+)-dependent digestive enzyme secretion (41). Here we demonstrate, using site-specific mutations, that Ca(2+)-sensitive phosphorylation at serine 136 modulates the accumulation of D52 at the plasma membrane within 2 min of cell stimulation. When expressed in Chinese hamster ovary CHO-K1 cells, D52 colocalized with adaptor protein AP-3, Rab27A, vesicle-associated membrane protein VAMP7, and lysosomal-associated membrane protein LAMP1, all of which are present in lysosome-like secretory organelles. Overexpression of D52 resulted in a marked accumulation of LAMP1 on the plasma membrane that was further enhanced following elevation of cellular Ca(2+). Strikingly, mutation of serine 136 to alanine abolished the Ca(2+)-stimulated accumulation of LAMP1 at the plasma membrane whereas phosphomimetic mutants constitutively induced LAMP1 plasma membrane accumulation independent of elevated Ca(2+). Identical results were obtained for endogenous D52 in normal rat kidney and HeLA cells, where both LAMP1 and D52 rapidly accumulated on the plasma membrane in response to elevated cellular Ca(2+). Finally, D52 induced the uptake of LAMP1 antibodies from the cell surface in accordance with both the level of D52 expression and phosphorylation at serine 136 demonstrating that D52 altered the plasma membrane recycling of LAMP1-associated secretory vesicles. These findings implicate both D52 expression and Ca(2+)-dependent phosphorylation at serine 136 in lysosomal membrane trafficking to and from the plasma membrane providing a novel Ca(2+)-sensitive pathway modulating the lysosome-like secretory pathway.

Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10 nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2′-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage – practically limited to base damage – has no satisfactory explanation.

Cell survival and chromosomal aberrations in CHO-K1 cells irradiated by carbon ions

Chinese hamster ovary CHO-K1 cells were exposed to high LET 12C-beam (LET: 830 keV/μm) in the dose range of 0–6 Gy and to 60Co irradiation and the RBE value was obtained. Effects of 12C-beam exposure on cell survival and chromosomal aberrations were calculated. The chromosomal aberration data were fitted with linear equation. The distribution of aberration in cells was examined with a standard u-test and used to evaluate the data according to Poisson probabilities. The variance to the mean ratio σ 2/ Y and the dispersion index ( u) were determined. Overdispersion was significant ( p<0.05) when the value of u exceeded 1.96.

Expression of tumor suppressor p53 facilitates DNA repair but not UV‐induced G2/M arrest or apoptosis in Chinese hamster ovary CHO‐K1 cells

Tumor suppressor p53 is an essential regulator in mammalian cellular responses to DNA damage including cell cycle arrest and apoptosis. Our study with Chinese hamster ovary CHO-K1 cells indicates that when p53 expression and its transactivation capacity was inhibited by siRNA, UVC-induced G2/M arrest or apoptosis were unaffected as revealed by flow cyotmetric analyses and other measurements. However, inhibition of p53 rendered the cells slower to repair UV-induced damages upon a plasmid as shown in host cell reactivation assay. Furthermore, the nuclear extract (NE) of p53 siRNA-treated cells was inactive to excise the UV-induced DNA adducts as analyzed by comet assay. Consistently, the immunodepletion of p53 also deprived the excision activity of the NE in the similar experiment. Thus, tumor suppressor p53 of CHO-K1 cells may facilitate removal of UV-induced DNA damages partly via its involvement in the repair mechanism.

Effects of exposure of CHO-K1 cells to a 10-T static magnetic field.

To evaluate whether exposure to strong static magnetic fields (SMFs), of up to 10 T, affects the growth and cycle distribution of and the micronucleus formation in monolayered Chinese hamster ovary CHO-K1 cells. The authors developed a system to expose cultured cells to strong SMFs immediately after the cells are seeded. Cell growth rate was evaluated according to cell number count. Cell cycle distribution experiments were performed by using flow cytometric analysis. In these experiments, the cells were exposed to SMFs for up to 4 days. The frequency of micronucleus formation with only SMF exposure at x-ray irradiation was analyzed at microscopic observation. Long-term exposure to a 10-T SMF for up to 4 days did not affect cell growth rate or cell cycle distribution. Exposure to SMFs alone did not affect micronucleus frequency. In x-ray-irradiated cells, exposure to a 1-T SMF did not affect micronucleus frequency, but exposure to a 10-T SMF resulted in a significant (P <.05) increase in micronucleus frequency. Strong (10-T) SMFs have no effect on cell growth, cell cycle distribution, or micronucleus frequency, but they may cause an increase in the micronucleus formation induced by 4-Gy x rays.

Expression of human metallothionein-III confers protection against serumfree exposure of stably transfected Chinese hamster ovary CHO-K1 cells

The cloned human metallothionein (MT)-III coding region was permanently transfected in Chinese hamster ovary (CHO-Kl) cells in order to investigate the growth regulatory effects of this brain-specific protein. Reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrated stable expression of MT-III mRNA in CHO-K1/MTIII cells. Whereas in the presence of serum no differences were observed between the cell growth of both CHO-K1/MT III and CHO-K1/pcDNA3-plasmid transfected cells, cell survival was attenuated differently in serum-free medium. CHO-K1/MT III cells were more resistant (40 ± 8%) to serum deprivation than pcDNA3-plasmid transfected cells. Recovery of cell growth for both cell lines was obtained by supplementing serum (0.25%), although with a different growth rate; half-maximal 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-conversion was observed between 5.1–5.7 and 4.2–4.5 days for CHO-K1/pcDNA3 and CHO-K1/MT-III cells, respectively. These results suggest a protective role for cloned human MT-III, besides its reported inhibitory and stimulatory effects on cell survival.

Characterization of the endogenous ADP-ribosylation of wild-type and mutant elongation factor 2 in eukaryotic cells.

Anti-[ADP-ribosylated elongation factor 2 (EF-2)] antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Natl Acad. Sci. USA 86, 554-557]. This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, chinese hamster ovary CHO-K1 cells was further characterized. The time course of labelling of ADP-ribosylated EF-2 with [32P]orthophosphate was similar in pyBHK cells and in CHO-K1 cells. The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2. The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2. When purified EF-2 from pyBHK cells was incubated with [carbonyl-14C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, [14C]NAD was generated. The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP-ribose acceptor site in EF-2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.

Reduced topoisomerase II and elevated α class glutathione S-transferase expression in a multidrug resistant CHO cell line highly cross-resistant to mitomycin C

We have isolated a multidrug-resistant derivative of Chinese hamster ovary CHO-K1 cells by exposure to progressively increasing concentrations of Adriamycin®. This cell line, designated CHO-Adr r, was 27-fold more resistant than the parental line to Adriamycin and showed similar degrees of cross-resistance to several other topoisomerase II (topo II) inhibitors, including mitoxantrone, daunomycin and etoposide. CHO-Adr r cells showed a lower (4-fold) level of cross-resistance to vincristine and colchicine, drugs associated with the multidrug-resistant phenotype. While CHO-Adr r cells showed no enhanced resistance to several mono- and bi-functional alkylating agents or to UV and ionizing radiation, they were greater than 80-fold resistant to mitomycin C (MMC). There was a 5-fold decreased level of daunomycin accumulation in CHO-Adr r cells compared to CHO-K1 cells and this was associated with increased drug efflux. The resistant cells had amplified multidrug resistance gene ( mdr) sequences and overexpressed ( mdr) mRNA. Verapamil was able to completely reverse Adriamycin resistance but reversal of MMC resistance was only partial, with residual 23-fold resistance. CHO-Adr r cells expressed a 4-fold reduced level of topo II protein but overexpressed an α class (basic) glutathione S-transferase (GST). Analysis of cell hybrids showed that while the level of resistance to Adriamycin dropped by a factor of 3 in CHO-K1/CHO-Adr r hybrids compared to CHO-Adr r/CHO-Adr r hybrids, resistance to MMC dropped 10-fold. Thus, CHO-Adr r cells appear to exhibit simultaneously several different drug resistance mechanisms including MDR and GST overexpression, and topo II reduction.

The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells.

Insulin is the only hormone required for continued growth of Chinese hamster ovary CHO-K1 cells in the defined medium M-F12. When CHO-K1 cells are incubated in M-F12 without insulin for 48-72 hr, the cells accumulate in G1. In response to physiological concentrations of insulin an 18-fold increase in rate of DNA synthesis occurs due to cells entering S phase after an 8- to 10-hr lag; cell division begins after 24 hr. The inhibitory effect of actinomycin D and 5,6-dichlorobenzimidazole riboside indicates that RNA synthesis is required for progression to S phase. CHO-K1 cells possess insulin receptors, and the insulin effect results from insulin binding to its own receptor: (i) Binding occurs at physiological insulin concentrations with a half-maximal stimulation at approximately 14 ng/ml. (ii) At insulin concentrations used, insulin-like growth factor I and II (IGF-I and IGF-II) have little or no effect. (iii) Scatchard analysis of 125I-labeled insulin binding shows the curvilinear response typical of insulin. (iv) The Kd for the so-called high-affinity binding site and the Ke are characteristic of the insulin receptor. (v) At the minimal insulin concentrations that stimulate growth, IGF-I and IGF-II compete poorly with insulin for insulin binding, insulin competes poorly with IGF-I for IGF-I binding, and affinity labeling with 125I-labeled insulin identifies a polypeptide (Mr = 125,000) typical of the alpha subunit of the insulin receptor.