Normal Hamster Cells Research Articles

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.

Asperaculanes A and B, two sesquiterpenoids from the fungus Aspergillus aculeatus.

Six sesquiterpenoids 1–6, including two new ones, an ent-daucane-type sesquiterpenoid, asperaculane A (1), and a nordaucane one, asperaculane B (2), and four known nordaucane derivatives, aculenes A–D 3–6, together with the known secalonic acid D (7), were isolated from a fermentation culture of the fungus Aspergillus aculeatus. Their structures and absolute configurations were established by analyses of their spectroscopic data, including 1D and 2D-NMR spectra, HR-ESIMS, electronic circular dichroism (ECD) data, and quantum chemical calculations. These metabolites were evaluated for in vitro cytotoxic activity against two cell lines, human cancer cell lines (HeLa) and one normal hamster cell line (CHO).

Semisynthesis and in vitro cytotoxic evaluation of new analogues of 1-O-acetylbritannilactone, a sesquiterpene from Inula britannica

Semisynthetic analogues of the natural product 1-O-acetylbritannilactone (ABL), a sesquiterpene isolated from the medicinal plant Inula britannica, have been prepared and exhibited significant in vitro cytotoxic activities against four cell lines including three human cancer cell lines (HCT116, HEp-2 and HeLa) and one normal hamster cell line (CHO). Structure–activity relationships indicate that esterification of 6-OH (enhanced lipophilicity) and α-methylene-γ-lactone functionalities play important roles in conferring cytotoxicity. Among the tested compounds, 14 bearing a lauroyl group (12C) at the 6-OH position displayed most potent in vitro cytotoxic activity, with IC50 values between 2.91 and 6.78 μM, comparable to the positive control etoposide (VP-16, IC50 values between 2.13 and 4.79 μM). Moreover, the compound 14 triggered remarkable apoptosis at a low concentration, and induced cell cycle arrest in G2/M phase in HCT116 cells. The biological assays conducted with normal cells (CHO) revealed that all the synthetic compounds are no selective against cancer cell lines tested.

Horizontal transmission and retention of malignancy, as well as functional human genes, after spontaneous fusion of human glioblastoma and hamster host cells in vivo

Cell fusion in vitro has been used to study cancer, gene mapping and regulation, and the production of antibodies via hybridomas. However, in-vivo heterosynkaryon formation by cell-cell fusion has received less attention. This investigation describes the spontaneous fusion of a human glioblastoma with normal hamster cells after xenogeneic transplantation, resulting in malignant cells that express both human and hamster genes and gene products, and retention of glioblastoma traits with an enhanced ability to metastasize. Three of 7 human genes found showed translation of their proteins during serial propagation in vivo or in vitro for years; namely, CD74, CXCR4 and PLAGL2, each implicated with malignancy or glioblastoma. This supports the thesis that genetic hybridization of cancer and normal cells can transmit malignancy and also, as first described herein, regulatory genes involved in the tumor's organotypic morphology. Evidence also is increasing that even cell-free human cancer DNA can induce malignancy and transfer genetic information to normal cells. Hence, we posit that the transfer of genetic information between tumor and stromal cells, whether by cell-cell fusion or other mechanisms, is implicated in the progression of malignancy, and may further define the crosstalk between cancer cells and their stromal neighbors.

Heat induces γH2AX foci formation in mammalian cells

H2AX is a histone variant which is present and ubiquitously distributed throughout the genome. An immunocytochemical assay using antibodies capable of recognizing histone H2AX phosphorylated at serine 139 (γH2AX) is very sensitive and is a specific indicator for the existence of a DNA double strand break. Although heat stress has been reported to induce the formation of γH2AX foci, no γH2AX foci formation was observed in several mammalian cell lines after heat shock. Since this was in contrast to earlier reports, the work described here was intended to verify that heat-induced γH2AX foci do form in mammalian cell lines other than the cell lines used in earlier reports concerning γH2AX foci formation. The cell lines used in this work includes cell lines with differing p53-gene status (H1299, H1299/ neo, H1299/m p53 and H1299/wt p53 cells), various cancer cell lines (HeLa, HepG2, U2-OS cells), normal human cells (HEK-293 and AG1522), and cell lines established from other species (MEF normal mouse cells and CHL normal Chinese hamster cells). Exponentially growing cells were exposed to heat shock (42 °C for 6 h or 45.5 °C for 20 min) or to X-rays (3 Gy). The presence of γH2AX was examined with immunocytochemistry and flow cytometry. Induction of γH2AX foci formation was observed in all of the mammalian cell lines used here after heat-treatment as well as after X-irradiation. However, the intensity of γH2AX was different in the different cell lines used. These results confirm that heat can induce γH2AX foci formation in many mammalian cell lines.

Sugar Transport Regulation: Comparative Characterization of the Effect of NADH CoQ Reductase Deficiency in Two Cell Culture Systems

Abstract. In this report, we have characterized the upregulation of glucose transport in two different respiration-deficient fibroblast cell cultures. We have demonstrated that glucose transport increases in respiration-deficient cells as measured by 2 deoxy D-glucose transport and is readily observed in both the WG750 human and G14 Chinese hamster fibroblast respiration-deficient cell lines when compared with the MCH55 normal human and V79 parental Chinese hamster cell lines, respectively. Using subcellular fractionation techniques, the GLUT 1 glucose transporter was found located predominantly in the plasma membrane–enriched fraction of the human and hamster cell lines. In human cells, the expression of the GLUT 1 glucose transporter was elevated three-fold in the plasma membrane–enriched fraction of the WG750 respiration-deficient mutant cells. In the Chinese hamster cell lines, the respiration-deficient G14 cells exhibited no such GLUT 1 glucose transporter elevation in the plasma membrane–enriched fraction, yet expressed a >2-fold increase in glucose transport. Furthermore, the G14 cells had a similar content of GLUT 1 glucose transporter in the plasma membrane fraction when compared with the V79 parental cell line. Using Western blot analysis, the GLUT 1 glucose transporter in G14 cells exhibited a different mobility on a polyacrylamide gel when compared with the mobility of the GLUT 1 glucose transporter of the V79 cell line. This differential mobility of the glucose transporters in the hamster cells appeared to be related to glycosylation differences of the glucose transporters. Although normal human and hamster cell lines exhibited significant increases in insulin-stimulated sugar transport (P < 0.05), the two respective respiration-deficient cell lines exhibited no significant increases in insulin-stimulated sugar transport (P > 0.05). Additionally, the expression of the GLUT 1 mRNA in the human WG750 mutant cells was elevated when compared with GLUT 1 mRNA in normal cells. Insulin exposure significantly increased GLUT 1 mRNA in human cells (P < 0.05). No differences in the GLUT 1 mRNA were observed between both hamster cell lines. Thus, both respiration-deficient cell lines are insulin resistant (i.e., regarding their insulin-stimulated sugar transport). The respiration-deficient mutation results in an increased sugar transport in the human and hamster cells; however, the human cells adapt to the mutation by increasing their levels of GLUT 1 mRNA and eventually membrane-located glucose transporters. On the other hand, the hamster cells adapt by apparently modifying their glucose transporters' intrinsic activity via glycosylation. We feel that these cell systems can be effective models to study the multiple factors involved in sugar transport regulation in vertebrate cells.

Induction of gamma-H2AX Foci in Lymphocytes, Fibroblasts and Tumor Cells by Single and Pulsed High Dose Rate X-rays

Purpose and background: Variation in radiosensitivity of normal tissues is related to genetic susceptibility (Svensson et al. 2006). Persistence of gamma-H2AX foci after ionizing radiation is higher in DNA-DSB repair deficient cells than in normal cells. We investigated whether differences in number of ?-H2AX foci may be useful as a predictor of radiosensitivity. Materials and methods: Induction of gamma-H2AX foci was measured in various cell types: freshly isolated lymphocytes, fibroblast cells (AT+/+, AT+/-, AT-/-), DNA-DSB repair deficient and normal hamster cells and human tumour cells (lung carcinoma, SW-1573 and prostate cancer cell lines with different TP53 status: LNCaP: TP53 wt, PC3: TP53 null and DU145: TP53 mutant). A radiation dose of 2.4 Gy was given as a single dose or as pulsed doses of 100 mGy in 24 h (PDR: 240 pulses of 100mGy, 1 pulse every 6 min). The number of gamma-H2AX foci was determined 30 min and 24 h after single dose and directly after PDR. Results: Maximal number of γ-H2AX foci occurred after single dose at 30 min after irradiation without significant differences in numbers of foci between different cell lines. The number of gamma-H2AX foci was radiation dose dependent and linearly correlated with clonogenic survival. Both 24 h after a single dose and after PDR more remaining foci are observed in repair deficient cells than in normal cells. In addition the TP53 null cells have more remaining foci than the mutant and wt TP53 cells. Conclusions: The detection gamma-H2AX after in vitro irradiation of lymphocytes and cell lines is simple, reproducible and sensitive. The number of residual gamma-H2AX foci after single dose as well as after PDR enables us to distinguish cells with differences in DNA-DSB repair capacities. However, it remains to be established whether gamma-H2AX assay applied in lymphocytes can be used to predict for late normal tissue toxicity after radiotherapy. Sponsored by the Dutch Cancer Foundation UVA2008-4019

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.

Differential radioprotective effects of misoprostol in DNA repaircell proficient and-deficient or radiosensitive systems

The protective effects of misoprostol (MP), an analogue of prostaglandin E, on X-ray-induced chromosomal aber1 rations, were studied in normal or mutant Chinese hamster cell lines grown as spheroids in vitro and on cell-killing in stem-cell spermatogonia of a mutant (scid) mouse strain or its wild-type. The mutant hamster cell lines chosen for this purpose are known to be either hypersensitive to the killing effects of X-rays and/or deficient in the repair of DNA double-strand breaks. The scid mice are deficient in the repair of DNA double-strand breaks. The results show that MP manifests varying degrees of radioprotection in all these systems, but the magnitude of these effects in the mutants is markedly reduced compared to their respective wild-type counterparts. These findings suggest a link between ionizing radiation sensitivity, DNA double-strand break repair capability and MP-mediated radioprotection.

Absence of terminal telomeric FISH signals in chromosomes from immortal Chinese hamster cells.

In contrast to normal Chinese hamster cells, terminal telomeric signals were not detectable by fluorescence in situ hybridization (FISH) in their immortalized counterparts, even when a sensitive cooled CCD camera was used for acquiring FISH images. Since the telomeres were detectable by FISH in immortal human cells, this suggests that hamster and human cells might have different mechanisms of telomere processing.

Expression of nanomolar-affinity binding sites for melatonin in Syrian hamster RPMI 1846 melanoma cells

Pharmacological studies indicate that Syrian hamster melanoma (RPMI 1846) cells posses a melatonin binding site similar to that found in normal hamster cells. A high correlation was observed for a series of compounds between the K i in hamster hypothalamic membranes vs. RPMI 1846 membranes ( r = 0.94, slope = 0.93, P < 0.01, n = 14). Scatchard analysis of saturation binding of 2-[ 125I]-iodomelatonin to membranes (at 0°C) indicated: K d = 0.89 ± 0.08 nM, B max = 6.2 ± 2.9 fmol/mg protein ( n = 3). Melatonin did not alter basal or forskolin-stimulated adenylate cyclase activity in RPMI 1846 membranes or intact cells. Therefore, in contrast to the picomolar-affinity receptor for melatonin in the mammalian hypothalamus and pars tuberalis, the putative nanomolar-affinity receptor is not coupled to adenylate cyclase. The RPMI 1846 cell line provides a useful model system for further studies of signal transduction via the nanomolar-affinity site for melatonin.

A proliferation-related constraint on endogenous and interferon-induced 2-5A synthetase activity in normal and neoplastic Syrian hamster cells.

2-5A Synthetase is one of the most extensively characterized enzymes induced by interferon (IFN) and is the central enzyme in a pathway that may be involved in the control of cellular proliferation. We examined the activity of this enzyme in normal diploid Syrian hamster cells (FC13) and their neoplastically transformed derivatives (BP6T); the former cell strain possesses regulated proliferative control, while the latter cell line has escaped from this control. A significant threefold increase in 2-5A synthetase activity was observed in density-arrested versus proliferating FC13 cells, whereas endogenous enzyme activity was uniformly low in BP6T cultures. The increase in enzyme activity in FC13 cultures was not accompanied by the production of IFN at a detectable level, but was parallelled by an increase in the intracellular level of 2',5'-oligoadenylate. IFN treatment resulted in a differential induction of enzyme activity depending on the proliferative state of FC13 cells. After IFN treatment, BP6T cells and subconfluent FC13 cells responded similarly with a fivefold increase in enzyme activity, whereas confluent FC13 cells displayed only a 1.4-fold increase. 2-5A Synthetase enzyme activity reflected steady-state mRNA levels in BP6T and subconfluent FC13 cells. In contrast, a noncoordinate regulation of 2-5A synthetase mRNA expression and enzyme activity was detected in confluent FC13 cells, suggesting that posttranscriptional mechanisms may be involved. The different patterns of endogenous and IFN-induced 2-5A synthetase enzyme activity in FC13 and BP6T cells found in this comparative study may represent an alteration fundamental to the loss of proliferative control in transformed cells.

Phenotypic properties of herpesvirus-transformed cells with high tumorigenic and metastatic ability

Herpes simplex virus type 2-transformed hamster embryo fibroblasts (333-8-9 cells) produce increased plasminogen activator (PA) compared with normal hamster cells. These cells produce undifferentiated fibrosarcomas at the inoculation site in newborn hamsters, and metastasize to the lungs. Using a direct PA assay, in which 125I-labeled plasminogen is cleaved, the optimum pH and osmolarity for detection of the 333-8-9 extracellular PA were pH 8.9 and approximately 150 mOsmol. Secretion of enzyme did not vary significantly on a per cell basis over cell densities from 0.1 to 8.0 X 10(7) cells/T-75 cm2 flask. This assay demonstrates that the 333-8-9 cells produce at least 20-fold greater levels of PA than normal cell counterparts. Based on the molecular weight (50-58 kDa) of secreted 333-8-9 cells PA and lack of fibrin stimulation, we conclude that it is a urokinase type PA. Subclonal lines of the 333-8-9 cells, selected for an increased PA phenotype were stable in culture, more tumorigenic and probably more metastatic. Correlation of these two events was examined by passaging 333-8-9 cells in vivo to select for greater tumorigenic potential and then determining the production of PA by the in vivo-derived sublines. The metastatic potential of the resulting cells was heterogeneous. Increased PA production upon increased passage in vivo did not always occur, whether the cells were passaged as subcutaneous tumors or as ascites tumors. Thus, while enzyme production correlated with tumorigenicity when selecting cells for an increased protease phenotype, this correlation was not observed when selecting for in vivo tumorigenicity. The results suggest that increased ability to make PA represents only one of multiple selective advantages for tumor growth.

Strand specificity for mutation induction in normal and repair-deficient UV-sensitive V79 Chinese hamster cells

Strand specificity for mutation induction in normal and repair-deficient UV-sensitive V79 Chinese hamster cells

Longterm survival of normal, diploid Syrian hamster cells in tumors induced by transfection with v-Ha- ras and v- myc oncogenes

Tumors were induced following transfection of normal, diploid hamster embryo cells with plasmids containing the viral Harvey ras and viral myc oncogenes. Direct cytogenetic studies of the tumors performed at 3–7 weeks after injection of the transfected hamster cells into nude mice revealed that 100% of the hamster cells were aneuploid and no detectable diploid cells in mitosis were observed. However, when tumor explants were cultured in vitro, diploid Syrian hamster cells were frequently detected at early passages. The percentage of diploid hamster cells in the cultures varied from 2 to 94% at the first passage. After several passages in vitro, only aneuploid hamster cells were observed. The diploid hamster cells in culture had a flat morphology and senesced. The aneuploid cells in the cultures were readily cloned and formed tumors after reinjection in nude mice. Reconstruction experiments consisting of injecting cloned, aneuploid tumor cells mixed with 6 × 10 6 normal Syrian hamster embryo cells were performed. Cell cultures derived from these mixed tumors contained both normal and aneuploid hamster cells indicating that normal cells survived in vivo during the growth of the tumor cells for up to 4 weeks. During this period, some normal cells transplanted in vivo did not die or terminally differentiate. Since normal cells can persist in vivo, tumor-derived cell cultures are mixed populations and caution should be exercised in interpreting quantitative molecular or cellular studies of these uncloned populations.

Human repair gene restores normal pattern of preferential DNA repair in repair defective CHO cells.

The pattern of preferential DNA repair of UV-induced pyrimidine dimers was studied in repair-deficient Chinese hamster ovary (CHO) cells transfected with the human excision repair gene, ERCC-1. Repair efficiency was measured in the active dihydrofolate reductase (DHFR) gene and in its flanking, non-transcribed sequences in three cell lines: Wild type CHO cells, a UV-sensitive excision deficient CHO mutant, and the transfected line of the mutant carrying the expressed ERCC-1 gene. The CHO cells transformed with the human ERCC-1 gene repaired the active DHFR gene much more efficiently than the non-transcribed sequences, a pattern similar to that seen in wild type CHO cells. This pattern differs from that previously reported in CHO cells transfected with the denV gene of bacteriophage T4, in which both active and non-transcribed DNA sequences were efficiently repaired (Bohr and Hanawalt, Carcinogenesis 8: 1333-1336, 1987). The ERCC-1 gene product may specifically substitute for the repair enzyme present in normal hamster cells while the denV product, T4 endonuclease V, does not be appear to be constrained in its access to inactive chromatin.

Potentially Lethal Radiation Damage Repair and Its Inhibition by Hyperthermia in Normal Hamster Cells, Mouse Cells, and Transformed Mouse Cells

The capacity of plateau-phase Chinese hamster V79 and normal and transformed C3H-10T1/2 cells for repair of potentially lethal radiation damage (PLD) was evaluated for cells irradiated alone or given combined treatments of heat and radiation. The data show that all cell lines tested could repair PLD and that transformation to the tumorigenic state may reduce the capacity to repair PLD, especially if cells are evaluated at equal survival levels. Hyperthermia treatments before irradiation produced less sensitization than treatments after irradiation. In addition, hyperthermia treatment led to the inhibition of cellular capacity to repair PLD. This effect was the greatest for cells heated after irradiation, and repair of PLD could be completely eliminated. Several temperature isodose heat treatments were evaluated, and the lower temperature heat treatments were more effective in the inhibition of PLD than the higher temperature heat treatments; this is consistent with earlier results indicating temperature dependence in thermal radiosensitization (S. A. Sapareto et al., Int. J. Radiat. Oncol. Biol. Phys. 5, 343-347 (1979)).

Correction of excision repair in xeroderma pigmentosum by hamster chromosome fragments involves both classes of pyrimidine dimers.

The ultraviolet light-sensitive phenotype of xeroderma pigmentosum (XP) has been corrected by the incorporation into XP cells of small chromosome fragments from Chinese hamster ovary cells. Like normal human and hamster cells, these XP-hamster hybrids are able to excise both of the photoproducts produced by ultraviolet light: cyclobutane pyrimidine dimers and the minor photoproduct, (6-4) pyrimidine-pyrimidone dimers. This excision capacity contrasts with that of an XP revertant, of the same cell line used in this study, which is able to excise only the (6-4) photoproducts. The excision defect of XP has been fully corrected in the hybrids; therefore, the small hamster chromosome fragments they contain should carry the gene for complementation group A of XP.

Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells.

Comparison by subtractive hybridization of mRNAs revealed a moderately abundant message in highly tumorigenic CHEF/16 cells present at very low levels in closely related nontumorigenic CHEF/18 cells. After cloning and sequencing the corresponding cDNA, computer comparison showed closest homology with the human connective tissue-activating peptide III (CTAP III). The human tumor cell cDNA hybridizing with the Chinese hamster clone was isolated, sequenced, and found to have closer similarity to the Chinese hamster gene than to CTAP III. Thus, the cloned cDNAs from Chinese hamster and human cells represent a different gene, named gro. Studies of its transcriptional regulation have shown that expression is tightly regulated by growth status in normal Chinese hamster and human cells and relaxed in the tumorigenic cells so far examined.

The oncogene of BAV-3 as a mutagen.

We studied the mutagenic and carcinogenic effects on mammalian cells of two EcoRI DNA fragments of bovine adenovirus type3 (BAV-3) integrated into the pBR325 plasmid. Fragment D located between 3.6 and 19.7 map units, contains the viral oncogene, fragment C, located between 44.3 and 63.7 map units, has no oncogenic activity. The BAV-3 oncogene was shown to increase significantly the frequency of 6-mercaptopurine (6MP)-resistant mutants in Chinese hamster calls. Fragment C, pBR325 without viral sequences and DNA from normal Syrian hamster cells did not have any mutagenic effect. We also looked at the combined action of the viral DNA fragments and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which enhances the transforming effect of carcinogens. TPA was shown to increase the mutant yield on exposure to the viral oncogene but not to induce mutagenic activity in those types of DNA that are unable to transform cells. Probably TPA does not affect the initiation of the mutation process, but acts on later stages just as it affects carcinogenic activity. Thus the results obtained confirm the existence of parallelism between the mutagenic and transforming effects of viral DNA and show that both activities are mapped in the same region of viral DNA - its oncogene.