Hamster Cell Line Research Articles

Abstract 5914: The porcine model for onocolytic adenovirus-based therapy

Abstract Oncolytic adenoviruses (Ad) are promising tools in the development of cancer therapeutics. A majority of Ad-based therapies utilize serotypes of species C, with Adenovirus type 5 (Ad5) being the most commonly employed. Previously, clinical trials have demonstrated the low efficiency of Ad5 vectors, mainly due to absence of the Ad5 primary receptor (Coxsackie Adenovirus Receptor, CAR) in cancer cells. Engineering Ad vectors utilizing the species B (Ad3, Ad35, Ad11) receptors have greatly improved the oncolytic potential of Ad-based therapies. However, the lack of a viable animal model has impeded clinical translation of these tropism-modified vectors. Mouse models are insufficient because Ad does not replicate in murine tissue. Non-human apes are not feasible due to availability and cost. Cotton rats and Syrian hamsters, although permissive of Ad5 replication, are not suitable for Ad3-, Ad35-, and Ad11-retargeted vectors due to the lack of species B primary receptors (CD46 and desmoglein 2) in rodent systems. In this study, we explored pigs as a model to study performance of the group B oncolytic adenoviruses by employing the fiber-modified Ad5/Ad3 chimeric vector. As a control, the Ad5 fiber-unmodified virus was used. First, we demonstrated the ability of swine cell lines to support replication of both Ad5 and Ad5/Ad3. Second, we analyzed binding, gene transfer, cytolytic, and replication ability of Ad5 and Ad5/Ad3 in various non-human cell lines (swine, hamster, murine, rat, bovine, canine). Our data confirmed that while hamster cell lines (HP1 and HapT1) were able to support binding and replication of Ad5-based vectors, they failed to support that with the Ad3-retargeted vectors. Additionally, we showed that among all tested cell lines, only porcine cells (PK15 and PTK75) were supportive of both binding and replication of the Ad3-retargeted virus. Of note, Ad5/Ad3 outperformed Ad5 in its cytolytic effect in porcine cell lines. These in vitro results prompted evaluation of the vectors in vivo. Immunocompetent Yorkshire pigs were systemically injected with a single dose of Ad5- and Ad5/3- expressing luciferase (Luc) from the Ad E3 region. Quantitative PCR analyses of the primary organs collected 7 days post-infection revealed Ad5 and Ad5/3 viral DNA in the lungs and spleen. Replication-dependent Luc expression was also observed in these tissue samples suggesting active viral replication. The quantity of viral DNA in other tissue such as the kidneys, liver, and pancreas was negligible. The results of these in vitro and in vivo studies indicate that pigs are a promising model to assess unmodified and tropism-modified adenoviral vectors. Citation Format: Malavika Chandrashekar, Lisa Koodie, Michele Dunning, George Ruth, Richard Bianco, Masato Yamamoto, Julia Davydova. The porcine model for onocolytic adenovirus-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5914.

Abstract 261: Chemopreventive effects of flavonoids on pancreatic carcinogenesis

Abstract Although clinical therapies are being developed for combating pancreatic cancer, its prognosis remains dismal; consequently, chemopreventive strategies may be needed to manage this disease. Luteolin (Lut), a flavone contained in perilla, is one of the most effective antioxidant flavonoids and has demonstrated potential for the treatment of inflammatory and neoplastic disease. Hesperetin (Hes) is a flavone contained in citrus, and the Ohsaki cohort study showed that citrus consumption was correlated with reduced pancreatic cancer (RR=0.62, 95%CI=0.38-1.00) (Int.J.Cancer, 2010: Li.W). However, there are no studies dealing with their antineoplastic effects against pancreatic cancer. In the present study, to assess the chemopreventive effects of Lut and Hes for pancreatic carcinogenesis, 6 week-old syrian golden hamsters were subcutaneously injected with N-nitrsobis(2-oxopropyl)amine four times and then fed Quick Fat Diet (control, n=14) or Quick Fat Diets with Lut 100 ppm (n=13) or Hes 100 ppm, 1000 ppm (n=13,13) for 13 weeks. There were no differences in body weights or liver or kidney weights among the groups. Serum cholesterol was significantly decreased in the Lut group. The incidence and multiplicity of pancreatic cancer was significantly suppressed in the Lut group (Incidence/ Multiplicity: 23%/ 0.23±0.44; P<0.05), but not the Hes groups (100ppm: 54%/ 0.69±0.75, 1000ppm: 62%/ 0.77±0.73), compared to the controls (71%/ 0.93±0.73). The Ki67 labeling index in pancreastic intraepithelial neoplasias (PanINs) was significantly decreased by Lut, but not Hes. Therefore, we focused further investigation on Lut. In vitro, cell proliferation of both hamster (HPD1NR) and human (MiaPaCa, PANC1) pancreatic cancer cell lines was inhibited by Lut in a dose-dependent manner. Multi-blot analysis for phosphorylated proteins related to cell signaling revealed that phosphorylated forms of Stat3 (tyr705) were down-regulated by Lut in MiaPaCa and PANC1 cells. We confirmed them in a dose-dependent manner in the three pancreatic cell lines and also found that Lut down-regulated cyclin D1. In vivo, the nuclear positive proportion of pStat3 (tyr705) in both PanINs and adenocarcinomas was significantly decreased by Lut. Because pStat3 (tyr705) is regulated by cytokines such as Il6, we examined mRNA levels of several cytokines including Il6 in hamster pancreatic tissues. Lut decreased the mRNA expression of Tgfb, Tnfa, Il10, and Il6. Immunohistochemistry of alpha smooth muscle actin revealed that the positive area of activated pancreatic stellate cells, which can produce these cytokines in cancerous tissue, tended to be decreased by Lut. In conclusion, these results indicate that Lut, but not Hes, has potential as a chemopreventive agent for pancreatic cancer and inhibition of pStat3 signaling may be involved in its mechanism of action. Citation Format: Hiroyuki Kato, Aya Naiki, Shugo Suzuki, Yoriko Yamashita, Satoru Takahashi. Chemopreventive effects of flavonoids on pancreatic carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 261.

Effect of extraction Alkaloids from Zephyranthes candida induces cell death through destruction microtubules in (China hamster cell line)

Background: In eukaryotic cells the cytoskeleton network consists of three major structural elements, microtubules, microfilament,and intermediate filaments. Tubulin is the basic protein of the MTs, molecules of tubulin arranged in dimmers consisting of two forms, α- tubulin and β-tubulin. They are continuously changeable structures.Taxanes and vinca alkaloids are inhibitors MTC that destabilize microtubules, there by suppressing their dynamics which required for proper mitotic function and effectively blocking cell regulation progression resulting in cell death. Objective: To evaluate the capability of alkaloids taken away from leaves of Zephyranthes candidato divert the MTC network of (CHO), which is an aggressive metastasis cell line. Patients and Methods: In our experiments we used the mouse CHO cell line. This cell line was obtained from the Department of Biology,Faculty of Medicine, Wuhan University, China. The cells were grown in Dulbecco’s Minimal Essential Medium (DMEM) (PAA Laboratories GmbH, Linz, Austria), supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 Uml–1 penicillin, and 100 μg ml–1 streptomycin (PAA Laboratories, Austria) in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C.Zephyranthes candida crude alkaloid extract treated cells were cultivated over microscopic cover slips, washed three Times for 4 min with PBS (pH 6.9) and fixed by 3% paraformaldehydein PBS. The ability of alkaloid extract to inhibit CHO tumor cells growth in vivo was assessed; forty mice were injected subcutaneouslyin the right dorsal with this tumor cells, after the tumor developed to 8-1 mm2, and eliminating the animalsthat did not develop tumors, the animals were divided to three groups each one with ten individuals. Results: The extractor was capable of divert the MTC of the cells below realization after 1hr of in sinuation in a focus as little as possible 20 μg/ml. while DAPI staining used, the cells death was not check in this focus and time. The cell death have been spotted when the focus of the alkaloid reproducer raised up to 80 and 100 μg/ml through the aforesaid exposure time. The cells were able to improving there native MTC contraction after 12 hr of the alkaloid deletion. The excerpt focus of 1mg/Kg/Bw competently prevent CHO cell line cancer development in vivo to 97.14% after 3 weeks therapy contrast to natural control. Conclusion: The invasive species H22 cells offer changesin the preparation of their MTC at a focus of Zephyranthes candida leaf alkaloids excerpt as low as 20 μg/ml focus after 1hr of exposuretime.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Biologics: structural heterogeneity and immunogenicity.

In principle the whole human proteome is available for the generation of recombinant proteins and glycoproteins that may serve as drugs (biologics). Endogenous human proteins and glycoproteins are structurally heterogeneous but are recognized as self by the immune system; however, recombinant protein and glycoprotein molecules are necessarily produced in heterologous systems and may include structural variants that are non-self and potentially immunogenic. The addition of human type oligosaccharides may be critical to function while the addition of non-human sugar residues can render biologics immunogenic. A particular concern is the structure of oligosaccharides attached by the hamster and murine cell lines that provide the dominant production platform. Critical structure and function properties that contribute to optimization of therapeutic potential are illustrated through recombinant erythropoietin and antibody therapeutics.

A comparative genomic hybridization approach to study gene copy number variations among chinese hamster cell lines

Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc.

Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a Treated with Nuwiq®, a New Generation Recombinant FVIII of Human Origin

Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a Treated with Nuwiq®, a New Generation Recombinant FVIII of Human Origin

Design, synthesis and biological activity of novel molecules designed to target PARP and DNA

In order to enhance the cytotoxic potential of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a PARP targeting scaffold. A cell-based selectivity assay involving a BRCA2-deficient Chinese hamster cell line and its corresponding BRCA2 wild type transfectant, was used to predict the PARP targeting potential of the latter agents. The results showed that adding a DNA damaging function to the PARP inhibitors decreased but did not abrogate the selective targeting of the BRCA2-deficient cells. The DNA damaging moiety augmented the potency in BRCA2 deficient cells by 2–20 fold. The most selective dual PARP–DNA targeting agent 14b was found to possess dual DNA and PARP targeting properties.

Mutagenic properties of linuron and chlorbromuron evaluated by means of cytogenetic biomarkers in mammalian cell lines

Agricultural practices are usually supported by several chemical substances, such as herbicides. Linuron and chlorbromuron are phenylurea herbicides largely used to protect crops from weeds, blocking photosynthesis by inhibition of the photosystem II complex. The former, also commercially known as lorox or afalon, is selectively used to protect bean and French bean plants, fennels, and celeriacs; the second, commercially known as maloran, is selectively used for carrots, peas, potatoes, soy sprouts, and sunflowers. Considering the widespread use of herbicides and, more generally, pesticides, it is important to clarify their involvement on human health, one of them concerning the possible direct or indirect effect on the genome of exposed populations. Here, we show that these herbicides are endowed by mutagenic properties, as demonstrated by an increased number of chromosomal aberrations (CAs) in two exposed Chinese hamster cell lines derived from ovary and epithelial liver, respectively. This was also confirmed by sister chromatid exchange (SCE) and micronucleus (MN) assays. Our present and previously obtained data clearly indicate that phenylurea herbicides must be used with great caution, especially for agricultural workers who use large amounts of herbicides during their work, and particular attention should be given to residues of these herbicides and their involvement in environmental pollution.

Transferring Xenogenic Mitochondria Provides Neural Protection Against Ischemic Stress in Ischemic Rat Brains.

Transferring exogenous mitochondria has therapeutic effects on damaged heart, liver, and lung tissues. Whether this protective effect requires the symbiosis of exogenous mitochondria in host cells remains unknown. Here xenogenic mitochondria derived from a hamster cell line were applied to ischemic rat brains and rat primary cortical neurons. Isolated hamster mitochondria, either through local intracerebral or systemic intra-arterial injection, significantly restored the motor performance of brain-ischemic rats. The brain infarct area and neuronal cell death were both attenuated by the exogenous mitochondria. Although internalized mitochondria could be observed in neurons and astrocytes, the low efficacy of mitochondrial internalization could not completely account for the high rate of rescue of the treated neural cells. We further illustrated that disrupting electron transport or ATPase synthase in mitochondria significantly attenuated the protective effect, suggesting that intact respiratory activity is essential for the mitochondrial potency on neural protection. These results emphasize that nonsymbiotic extracellular mitochondria can provide an effective cell defense against acute injurious ischemic stress in the central nervous system.

410. Oncolytic Adenoviruses Armed with TNFa and IL-2 Induce Antitumor Immune Responses and Protection from Tumor Rechallenge

During the past few years, immune system-stimulating factors have emerged as promising means to treat malignant tumors. We aim to use cytokine-bearing oncolytic adenoviruses to improve and to extend the usage of adoptive T cell therapy, a treatment known to benefit a portion of melanoma and leukemia patients. Administered systemically or intratumorally the virus induces immune responses against the tumor by revealing tumor antigens, but immunostimulatory cytokines augment the effect further. We have shown that the most promising cytokines in this regard are interleukin (IL) -2 and Tumor Necrosis Factor alpha (TNFa). IL-2 is a common treatment for malignant melanoma and renal cell carcinoma, but systemic administration may lead to severe side effects. While IL-2 has a key role in recruiting and activating T cells, TNFa has prominent anti-immunosuppressive actions. Further, it directly promotes tumor cell death by apoptosis and necrosis. Armed oncolytic viruses accomplish local, long-lasting, high-level cytokine expression while systemic level remains low. Moreover, we have proven that adenoviruses enhance adoptive T cell therapy. In this study, we treated Syrian hamsters (Mesocricetus auratus) with Ad5/3-E2F-d24 virus bearing human IL-2, TNFa, or both in its E3 region. Hamster cell lines are semipermissive for 5/3-chimeric adenoviruses and produce active transgenes from these viruses. In addition, human IL-2 and TNFa are evidently active in hamsters. Hamster pancreatic cancer (HapT1) was implanted subcutaneously and treated once with TILs extracted from syngenic tumors, and with five viral injections. We saw synergy between unarmed virus and TILs, while the armed viruses turned out to be even more effective. When the cured animals were rechallenged with the same cancer cells, previous treatment with cytokine-armed viruses protected the animals against new tumors. Also, splenocytes derived from animals treated with cytokine-bearing viruses proliferated more actively ex vivo than the controls. To conclude, our data demonstrates the immunological benefit gained with viruses bearing IL-2 and TNFa, and further supports combining oncolytic viruses with adoptive cell transfer.

Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis.

Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNAMet in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.

Differential cell line susceptibility to the emerging Zika virus: implications for disease pathogenesis, non-vector-borne human transmission and animal reservoirs

Zika virus (ZIKV) is unique among human-pathogenic flaviviruses by its association with congenital anomalies and trans-placental and sexual human-to-human transmission. Although the pathogenesis of ZIKV-associated neurological complications has been reported in recent studies, key questions on the pathogenesis of the other clinical manifestations, non-vector-borne transmission and potential animal reservoirs of ZIKV remain unanswered. We systematically characterized the differential cell line susceptibility of 18 human and 15 nonhuman cell lines to two ZIKV isolates (human and primate) and dengue virus type 2 (DENV-2). Productive ZIKV replication (⩾2 log increase in viral load, ZIKV nonstructural protein-1 (NS1) protein expression and cytopathic effects (CPE)) was found in the placental (JEG-3), neuronal (SF268), muscle (RD), retinal (ARPE19), pulmonary (Hep-2 and HFL), colonic (Caco-2),and hepatic (Huh-7) cell lines. These findings helped to explain the trans-placental transmission and other clinical manifestations of ZIKV. Notably, the prostatic (LNCaP), testicular (833KE) and renal (HEK) cell lines showed increased ZIKV load and/or NS1 protein expression without inducing CPE, suggesting their potential roles in sexual transmission with persistent viral replication at these anatomical sites. Comparatively, none of the placental and genital tract cell lines allowed efficient DENV-2 replication. Among the nonhuman cell lines, nonhuman primate (Vero and LLC-MK2), pig (PK-15), rabbit (RK-13), hamster (BHK21) and chicken (DF-1) cell lines supported productive ZIKV replication. These animal species may be important reservoirs and/or potential animal models for ZIKV. The findings in our study help to explain the viral shedding pattern, transmission and pathogenesis of the rapidly disseminating ZIKV, and are useful for optimizing laboratory diagnostics and studies on the pathogenesis and counter-measures of ZIKV.Emerging Microbes & Infections (2016) 5, e93; doi:10.1038/emi.2016.99; published online 24 August 2016

Antigenotoxic, anti-photogenotoxic and antioxidant activities of natural naphthoquinone shikonin and acetylshikonin and Arnebia euchroma callus extracts evaluated by the umu-test and EPR method

The aim of this study was to evaluate the antigenotoxic and antioxidant potential of shikonin (SH), acetylshikonin (ACS) and Arnebia euchroma callus extract (EXT). The antigenotoxic activity was investigated by the umu-test as the inhibition of the SOS system induction caused by genotoxic chemical agents — 4-nitroquinoline oxide and 2-aminoanthracene. Moreover the ability of SH, ACS and EXT to prevent photogenotoxicity triggered by chlorpromazine under UVA irradiation was measured. The cytotoxicity of EXT toward V79 Chinese hamster cell line was additionally assessed. Shikonin and acetylshikonin had no effect on 4-NQO induced genotoxicity whereas EXT demonstrated an unclear effect. The protection against 2AA induced genotoxicity was observed for all tested substances. The highest protection was demonstrated for EXT with inhibition of 66%. SH and ACS reduced 2AA genotoxicity with inhibition of about 60%. Under UVA the strongest and dose-dependent activity was observed for EXT. Acetylshikonin was a weak anti-photogenotoxin whereas shikonin had no clear effect. EXT was highly cytotoxic toward the V79 cell line — the cells' morphology was affected seriously and apoptosis was impacted.The antioxidant activity of SH, ACS and EXT was studied by means of electron paramagnetic resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. All three samples exhibited radical scavenging properties.

DNA content of individual chromosomes of Chinese hamster normal karyotype and its permanent cell lines V-79 RJK and Verb-5

The genome size of the Chinese hamster Cricetulus griseus, absolute and relative DNA content of individual chromosomes in normal Chinese hamster karyotype and Chinese hamster cell lines V-79 RJK and Vebr-5 during long-term cultivation were determined with flow cytometry and microfluorimetry. It has been shown that the genome size of Chinese hamster males and females is 6.660 and 6.746 pg, respectively. DNA content in chromosomes of the cultivated cells drastically differed from that the normal Chinese hamster karyotype. It changed during the long-term cultivation of both cell lines. In certain chromosomes, the changes reached 20–25% (as a rule, increased). The level of DNA amplification in the p-arm of chromosome Z6 in cells Vebr-5 registered at the beginning of the experiment in the course of further cellular cultivation (over 20 years) remained stable. The data obtained suggest that the cell malignant transformation and cellular adaptation to the conditions in vitro resulted in the dramatic genome restructuring affected all chromosomes.

Insufficient sex description of cells supplied by commercial vendors.

evidence supporting sex as a biologically important variable in human and animal studies has been accumulating over recent decades ([1][1], [4][2], [6][3]–[8][4], [10][5]). As a result, the necessity of including female as well as male subjects in clinical studies has been widely recognized ([1][1

Antitumor effects of vitamin D analogs on hamster and mouse melanoma cell lines in relation to melanin pigmentation.

Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)2D3) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)2D3 and 25(OH)D3), and novel relatively non-calcemic ones (20(OH)D3, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner.

The major DNA repair pathway after both proton and carbon-ion radiation is NHEJ, but the HR pathway is more relevant in carbon ions.

The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays ((137)Cs), protons (200 MeV; 2.2 keV/μm) and C ions (290 MeV; 50 keV/μm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs(-/-) cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results suggest that the NHEJ pathway plays an important role in repairing DSBs induced by both clinical proton and C-ion beams. Furthermore, in C ions the HR pathway appears to be involved in the repair of DSBs to a greater extent compared to gamma rays and protons.

Evaluation of photodegradation, phototoxicity and photogenotoxicity of ofloxacin in ointments with sunscreens and in solutions

Fluoroquinolones are widely used anti-bacterial agents that are known to exhibit moderate to severe phototoxicity. Furthermore some of them reveal photogenotoxicity under UV irradiation. Incidence of side effects due to light exposure may be augmented, if the medicament is used topically. The main goal of this work was to compare the extent of photodegradation of ofloxacin in ointments with various excipients: hydrated or non-hydrated base and the addition of sunscreens: bisoctrizole (Tinosorb® M) and bemotrizinol (Tinosorb® S). The next goal of present work was the analysis of phototoxicity and photogenotoxicity of ofloxacin photodegradation products in tested ointments and in solutions with the umu-test, the test of mitotic gene conversion with Saccharomyces cerevisiae D7 and the micronucleus assay with V79 Chinese hamster cell line. At the same time an attempt was made to determinate the photodegradation products of ofloxacin in different unguents variants. We observed a significant photoprotective effect in ointment with Tinosorb® M. We did not evaluated relevant differences regarding the genotoxicity and toxicity of unguents. However, the pre-irradiated ofloxacin solutions in comparison to samples stored in the dark were significantly more genotoxic to bacteria, slightly increased the number of micronuclei in V79 cell line and were toxic to the yeast strain.

Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals

Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses.