A simplified procedure for the observation in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in mammalian monolayer cell cultures

Chinese hamster V-79 cells are widely used in short term screening for potential physical or chemical mutagens of the environment. A simplified version of the standard Giemsa protocol of Moorhead and the Feulgen plus Giemsa protocol of Wolff and Perry is given which permits the observations in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in the Petri dishes for the culture of the V-79 cells.     

The effect of synthetic polycation polyallylamine on adhesion and viability of CHL V-79 RJK Chinese hamster fibroblasts with different heat resistance

The effects of synthetic polycation polyallylamine (PAA) on adhesion of CHL V-79 RJK fibroblasts and CHL V-79 RJK40 cells resistant to 40 degrees C, and attachment to these cells to polycation immobilized on polystyrene surface were studied. We have also investigated the cytotoxicity of PAA. It was shown that cell adherence to polystyrene plastic coated with PAA was enhanced or decreased in dependence of the PAA concentration used for surface coating and did not depend on heat resistance of investigated cell lines. The effect of PAA on cell adhesion to uncoated polystyrene surface after cell exposure with PAA depended not only on the polycation concentration, but also on the extent of heat resistance of investigated cell lines. Pretreatment of CHL V-79 RJK cells with PAA at the nontoxic concentrations led to inhibition of cell adhesion, and no change in adhesive properties of thermoresistant cells was found under the same conditions. PAA was toxic for CHL V-79 RJK and CHL V-79 RJK40 cells only at concentrations of 100 microg/ml (MTT assay). PAA-induced acute toxicity was accompanied by necrotic-like cell damage. Possible mechanisms of the PAA effect on the behaviour of cells with different structural and metabolic characteristics that are due to the temperature of cell cultivation are discussed.     

Effect of nitric oxide donor (NaNO2) on the stability of non-transformed and malignant cells to ultraviolet and gamma-radiation

A decreased sensitivity of the Chinese hamster cells (line V-79) to gamma-radiation under the influence of nitric oxide induction was shown elsewhere. This effect is connected hypothetically with post-radiation reparation of DNA. The investigation of the nitric oxide donor effect on sensitivity of these to UV-radiation is of interest, because this radiation is an important ecological factor of the environment. The question of retention of nitric oxide positive effect on UV and gamma-radiation sensitivity in malignant HeLa cells is no less actual, because these cells significantly differ from normal cells of line V-79. We demonstrated that the donor of nitric oxide enhances stability of the Chinese hamster cells (line V-79) to UV-radiation, as well as to gamma-radiation independently of the time of cell incubation with sodium oxide donor before or after irradiation. The inefficiency of nitric oxide as a factor increasing UV-stability of cells was shown for malignant HeLa cells. A 1 h long incubation of these cells with NO-donor before gamma-irradiation decreased the number of chromosome aberrations, and conversely, the addition of this agent to the HeLa cell culture after gamma-irradiation did not change the radiostability. It may be inferred that distinctions in behaviour of nitric oxide in cultures of V-79 and HeLa cells using UV-radiation may be explained by transformation of the latter special features of their damage, and by the following reparation.     

Effect of synthetic polycation polyallylamine on adhesion and viability of CHL V-79 RJK Chinese hamster fibroblasts with various heat resistance

The effects of synthetic polycation polyallylamine (PAA) on the adhesion of CHL V-79 RJK fibroblasts and CHL V-79 RJK40 cells resistant to 40°C and the attachment of these cells to polycation immobilized on a polystyrene surface have been studied. We also investigated PAA cytotoxicity. It was shown that cell adhesion on polystyrene plastic coated with PAA depended on the PAA dose and did not depend on the heat resistance of the cells. The effect of PAA on cell adhesion to uncoated polystyrene surface after cell exposure to PAA depended not only on the polycation concentration but also on cell heat resistance. Pretreatment of cells with nontoxic concentrations of PAA inhibited CHL V-79 RJK cell adhesion and did not change adhesive properties of thermotolerant cells. PAA is toxic for CHL V-79 RJK and CHL V-79 RJK40 cells only at concentration of 100 μg/ml (MTT assay). PAA-induced acute toxicity was accompanied by necrotic-like cell death. Possible mechanisms of the PAA effect on the behavior of cells with different metabolic characteristics defined by heat resistance are discussed.     

Expression of the E. coli fpg gene in mammalian cells reduces the mutagenicity of gamma-rays.

The E. coli fpg gene encodes the formamido-pyrimidine-DNA-glycosylase (FPG protein) which specifically removes the formamido-pyrimidine and C8-oxoGuanine residues from gamma-irradiated DNA. The fpg gene was ligated in the psV2 vector and transfected into the Chinese hamster CHO and V-79 cells. The transfected cells expressed a formamido-pyrimidine-DNA-glycosylase activity 30 to 40-fold over the constitutive level. The resistance of CHO and V-79 cells to the lethal effect of gamma-rays was similar in control and transfected cells. Furthermore CHO cells expressing the fpg gene had the same resistance to the lethal effect of hydrogen peroxide as control cells. However, the sensitivity to the mutagenic effect of gamma-rays, measured as 6-thioguanine resistance, decreased both in CHO and V-79 transfected cells. Since the lethal effect of gamma-rays was not modified in cells overproducing the FPG protein, the results suggest that this protein protects the cells against the mutagenic lesions formed by ionizing radiations, and among them C8-oxoguanine.     

Decreased incidence of gap junctions between Chinese hamster V-79 cells upon exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate

Previous studies have shown that metabolic cooperation between Chinese hamster V-79 cells is inhibited by the tumor-promoting phorbol esters, but not by those phorbol esters inactive in tumor promotion. Metabolic cooperation is believed to be mediated by gap junctions. Therefore, in the present study we have used freeze-fracture and quantitative morphological techniques to examine the effect of the most potent tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of the inactive analog, 4α-phorbol-12,13-didecanoate (4α-PDD) on the frequency of gap-junctional contacts between V-79 cells. In both control and in 4α-PDD treated cultures the junctions were frequent, though rather small in size. In contrast, in the TPA-treated cultures, gap junctions were few, and the area of membrane occupied by gap junctions was reduced more than 20-fold from that found for controls. These results suggest that the TPA-induced inhibition of metabolic cooperation between V-79 cells is a consequence of a decrease in the number of gap junctions.     

Difference between diploid and aneuploid Chinese hamster cells in replication at mid-S-phase

Following partial synchronization of the heteroploid Chinese hamster cell line V-79 and of normal diploid lung fibroblasts of the Chinese hamster in culture, their DNA replication during S-phase was compared by means of a BrdU-incorporation/thymidine pulse technique and Hoechst-Giemsa differential staining of metaphase chromosomes. This comparison indirectly shows the S-phase of the heteroploid cells of V-79 to be 2 h shorter than the diploid cell S-phase. When the thymidine pulse is applied to diploid lung fibroblasts at mid-S-phase, differential staining colours metaphase chromosomes a pale blue. Performing the corresponding experiment with V-79 cells, neither a pale blue nor dark red staining is obtained, but rather an intermediate shade, showing prominently dark staining regions in parts. The pause in DNA synthesis observed at mid-S-phase of the diploid Chinese hamster lung fibroblasts seems to be omitted at mid-S-phase of the V-79 cells.     

DNA synthesis and cell survival after X-irradiation of mammalian cells treated with caffeine or adenine.

The expression of the transient depression in the rate of DNA synthesis normally observed after exposure of randomly-dividing Chinese hamster V-79 or Chinese hamster CHO cells to ionizing radiation can be postponed or diminished by a post-irradiation treatment with 1.0 to 1.0 mM adenine or 1.5 mM caffeine. Caffeine may exert its effect by creating additional sites for replication in irradiated cells. Cells treated with caffeine or adenine for 2 or 4 hours after exposure to 3000 rad of 300 kVp X-rays exhibit depressed synthesis only after the removal of caffeine or adenine. These alterations in the timing of the X-ray-induced depression of the rate of DNA synthesis have no effect on X-ray-induced cell killing. Although a 4 hour post-irradiation treatment of randomly-dividing Chinese hamster V-79 cells with 1.0 or 2.0 mM caffeine potentiates X-ray-induced cell killing, this reduction in survival is due primarily to effects on cells in S-phase.     

NO-dependent pathway of the modifying of the cellular sensitivity to gamma- and neutron irradiation

The modifying effect of L-NAME, the NO-synthase inhibitor and D-NAME, the inactive enantiomer was investigated in human carcinoma cells (HeLa) and Chinese hamster fibroblasts (V-79) exposed to different doses of gamma-rays and 0.85 MeV neutrons. We estimated the level of the chromosome aberrations manifested as the bridges and fragments in anaphases. Radioprotective effect of L-NAME showed the inverse dependence on the exposure dose and at low doses (1 Gy) it was higher in the V-79 cells, than in the HeLa cells. However, at high doses (3, 4, 6 Gy) the efficiency of L-NAME for these cellular lines was almost equal (DFR = 2). The modifying effect of L-NAME was almost equal for gamma-irradiation and neutrons, although the exposure of V-79 cells to neutrons induced more the asymmetric chromosome aberrations (RBE = 4). The D-NAME had no effect on the level of the radiation-induced chromosome aberrations, although D-NAME treatment of cells increased the chromatin condensation, as well as L-NAME. The counteractive condensation does not play the major role in the radioprotective effect of L-NAME. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the inducible NO-synthase.     

Lack of effect of glutathione depletion on cytotoxicity, mutagenicity and DNA damage produced by doxorubicin in cultured cells

Since endogenous glutathione (GSH), the main non-protein intracellular thiol compound, is known to provide protection against reactive radical species, its depletion by diethylmaleate (DEM) was used to assess the role of free radical formation mediated by doxorubicin in DNA damage, cytotoxicity and mutagenicity of the anthracycline. Subtoxic concentrations of DEM that produced up to 75% depletion of GSH did not increase doxorubicin cytotoxicity in a variety of cell lines, including Chinese hamster ovary (CHO) and lung (V-79) cells, LoVo human carcinoma cells and P388 murine leukemia cells. Similarly, the number of doxorubicin-induced DNA single strand breaks in CHO cells and the mutation frequency in V-79 cells were not affected by GSH depletion. The results obtained suggest that mechanisms other than free radical formation are responsible for DNA damage, cytotoxicity and mutagenicity of anthracyclines.     

Effect of ectopic superexperssion of the anti-apoptotic gene bcl-2 on the level and character of karyotypic instability in the CHLL V-79 RJK Chinese hamster cell line

Karyotypic destabilization in cells of Chinese hamster fibroblasts CHL V-79 RJK with ectopically overexpressed antiapoptotical human bcl-2 gene from pSFFV-bcl-2 vector has been analysed. Analysis of G-banded metaphase chromosomes from 4 clones with different levels of bcl-2 expression revealed an increased level of chromosomal instability in bcl-2-transfected cells. Besides, an increased percentage of aneu- and polyploid cells and high level of cells with different chromosomal aberrations was observed. The degree of karyotypic instability positively correlated with the level of bcl-2 expression in bcl-2-transfected cells. Cells of a clone with the highest bcl-2 expression at the 13th passage of cultivation displayed an almost 100% polyploidization and the presence of specific aberrations and a tricentric marker chromosome. Selection of cells with non-random specific chromosome changes was observed in pSFFV-bcl-2-transfected CHL V-79 RJK cells in the process of their long-term cultivation. By contrast, cells of the parental cell line, as well as the control pSFFV-neo transfectants, displayed a stable karyotype throughout the long period of cultivation. It is important that the presence of morphological markers of gene amplifications--DOO, DM, MH--was observed in bcl-2-transfected cells. These findings suggest that the overexpression of antiapoptotic human bcl-2 gene may result in destabilization of the karyotype structure in cells of Chinese hamster fibroblasts CHL V-79 RJK. The character and level of destabilization correlate with the level of ectopic overexpression of this gene.     

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during proliferation of Chinese hamster V-79 and human HeLaS-3 cells

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during cell proliferation were examined in Chinese hamster V-79 (V-79) cells (normal cells) and human HeLaS-3 (HeLaS-3) cells (malignant cells). For V-79 cells, the Mg2+-ATPase activity per cell (pmol Pi/h/cell) in the confluent phase was higher than that in the logarithmically growing (log) phase. The amount of microsomal protein per cell was also high in the confluent phase. Specific activities (mumol Pi/h/mg protein) of Na+, K+-, Mg2+- and Ca2+-ATPase were significantly lower in the confluent phase than in the log phase. For HeLaS-3 cells, an increase in Ca2+-ATPase activity per cell was observed. The amount of microsomal protein per cell did not change between the log and confluent phase. The specific activity of Ca2+-ATPase in the confluent phase was also markedly higher than in the log phase. The relation between changes in ATPase activities and cell proliferation is discussed.     

For the reproductive death of a cell, damage to a single chromosome is sufficient

The Chinese hamster cells V-79 were treated with BUdR during one cell cycle; after that the cells were grown in the medium without BUdR and were irradiated by longwave-UV-light at different time. The cell survival after photolysis was compared with the percentage of metaphase plates with different number of chromosomes containing BUdR. It is concluded that for cell inactivation the presence of only one destroyed chromosome (or its part) is enough.     

Mutant cell line demonstrating a block in insulin and insulin-like growth factor type 1 (IGF-1) induced mitogenesis

Recently, we have isolated a Chinese hamster cell variant (IV-A1-j) resistant to an insulin-diphtheria-A chain toxic conjugate (Leckett and Germinario: Cytotechnology [in press]. This cell line exhibited a decreased level of insulin binding, but normal growth in serum-containing medium when compared to the parental cell line (V-79). In this paper we further demonstrate that while IV-A1-j cells are capable of growing in serum-containing medium, they are insensitive to the mitogenic actions of either insulin or IGF-1. In contrast, epidermal growth factor (EGF) and/or α-thrombin (THR) generate a mitogenic effect in IV-A1-j cells comparable to that observed in the parental V-79 cells. The combination of EGF and/or THR with either insulin or IGF-1 results in an increase in V-79 cell growth above EGF and/or THR alone. On the other hand, insulin or IGF-1 in the presence of other mitogens did not stimulate further growth in IV-A1-j cells. While insulin binding was lower in IV-A1-j cells, internalization of ¹²⁵I-insulin was not different in the two cell types. Additionally, insulin-stimulated glycogen synthesis and protein synthesis were not different in the two cell types. These observations are consistent with insulin and IGF-1 sharing a mitogenic signalling pathway in Chinese hamster fibroblasts and that this pathway is distinct from other growth factor signalling pathways. The fact that this pathway is defective in the IV-A1-j cell line indicates the potential usefulness of these cells in identifying a key step(s) in the insulin (IGF-1) mitogenic pathway. © 1993 Wiley-Liss, Inc.     

A Comparison of Survival and Repair of Uv-Induced DNA Damage in Cultured Insect VERSUS Mammalian Cells

Survival and unscheduled DNA synthesis (UDS) were measured in a cultured insect cell line, TN-368, and a cultured mammalian cell line, V-79-4, following exposure to several fluences of ultraviolet light. TN-368 cells were approximately seven times more resistant to the lethal effects of UV than V-79 cells, as determined by colony formation. The amount of UDS per unit amount of DNA is about the same in both cell types 4 hr after 10–50 J/m² UV irradiations.     

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.     

Characteristics of the binding of labeled fluoromisonidazole in cells in vitro

Radiolabeled fluoromisonidazole (FMISO) is being investigated as an imaging agent for hypoxia in tumors and nonmalignant tissues in myocardial infarct or stroke. In this study in vitro cell cultures were used to characterize the oxygen dependency of FMISO uptake and to examine other modifying factors. The uptake of [3H]FMISO was measured in four cell lines in vitro: V-79, EMT-6(UW), RIF-1, and CaOs-1. The modifying effects of different O2 levels as well as cell growth state and concentration of glucose and nonprotein sulfhydryls were examined. In these cell types an O2 level between 720 and 2300 ppm inhibited FMISO binding by 50%, relative to binding under anoxic conditions. These values bracket the O2 level which confers full radiobiologic hypoxia, about 1000 ppm. Some bound label was released from cells in the first 1 to 3 h after a 3-h anoxic labeling with [3H]FMISO, but this does not represent tritium loss from the parent molecule. Cells from unfed plateau-phase cultures took up less [3H]FMISO than did exponentially growing cells incubated at comparable O2 levels. Reducing glucose to 1/10 or 1/100 of the usual concentration in medium had little effect on binding of micromolar levels of FMISO, except in V-79 cells, where reduced glucose levels were associated with increased FMISO accumulation. Adding cysteamine to the culture medium moderately increased FMISO uptake. We conclude that cell growth state, glucose, and nonprotein sulfhydryl concentrations affect FMISO binding, albeit less than varying O2 levels: anoxic/oxic binding ratios vary from 12.6 to 28 for the four cell types examined. Nonetheless these factors must be considered in evaluating the oxygen-dependent binding of this nitroimidazole in tumors or tissues.     

In situ oxygen consumption rates of cells in V-79 multicellular spheroids during growth

The rate of consumption of oxygen by V-79 cells in multicellular spheroids was measured as a function of the spheroid diameter. In situ consumption was equal to that of exponentially growing cells for spheroids less than 200 micron in diameter. The rate of oxygen consumption decreased for cells in spheroids between 200 and 400 micron diameter to a value one-fourth the initial, then remained constant with further spheroid growth. Comparison of consumption rates for spheroid-derived cells before and after dissociation from the spheroid structure indicated that the spheroid microenvironment accounted for only 20% of the change in oxygen consumption rate. Cell-cell contact, cell packing, and cell volume were not critical parameters. Plateau-phase cells had a fivefold lower rate of oxygen consumption than exponential cells, and it is postulated that the spheroid quiescent cell population accounts for a large part of the intrinsic alteration in oxygen consumption of cells in spheroids. Some other mechanism must be involved in the regulation of cellular oxygen consumption in V-79 spheroids to account for the remainder of the reduction observed in this system.     

Karyotype stability of 2 continuous Chinese hamster cell lines--CHO-K1 and V-79

Karyotypes of two continuous Chinese hamster cell lines CHO-K1 and V-79 were studied by G-banding and silver staining. Modal chromosome numbers were 20 and 21, respectively. Both the lines were characterized with a high degree of karyotype stability and constant ratio of normal and marker chromosomes. Nulli- and monosomy were recorded for 9 chromosome pairs in CHO-K1, and 8 pairs in V-79 cell lines. Modal numbers of Ag-positive NOR were 4 in CHO-K1 and 5 in V-79. A definition of the origin of the majority of marker chromosomes in both the lines (11 and 10, respectively) made it possible to establish the exact chromosome content of each cell and to determine the generalized reconstructed karyotypes of cell lines. We established the retention of diploid chromosome set of all the autosomes, the true monosomy for one X-chromosome in both the lines, and the constant extracopying of a short arm of chromosome 3 in the V-79 cell line.     

A comparative investigation of the antimitotic action of 2-mercaptoethanol and colcemid on V-79 Chinese hamster cells

Abstract. The current study was performed to characterize the antimitotic action of 2-mercaptoethanol (MET) on mammalian cells.
At concentrations of 2.5 × 10^-2 M, MET arrests V-79 Chinese hamster cells in metaphase. Smaller concentrations (from 5 × 10^-3 M) only produce a mitotic block after several hours, only arresting those mitoses which have gone through one cell cycle in the presence of MET. The accumulation of mitoses by MET is smaller in comparison with colcemid, explained by an effect reducing the number of cells which enter mitosis. In contrast to colcemid, MET-concentrations which do not lead to a mitotic block cause a delay in proliferation. It was shown, by means of the BUdR-labelling method that cells in the presence of colcemid concentrations which arrest mitosis again enter interphase and become polyploid, whereas MET leads to an irreversible arrest of mitosis and does not produce polyploidy in V-79 cells.