Identification of a chromosome that controls malignancy in Chinese hamster cells.

A chromosome that controls malignancy in Chinese hamster cells has been identified by analysis of the Giemsa banding pattern of a malignant cell line transformed by simian virus 40 (SV40), non-malignant revertants from this line, segregants from the revertants that were again malignant and a cell line transformed by methylcholanthrene. The malignant cell line transformed by SV40 was near diploid and had gained additional material of chromosome 3. Revertants with a suppression of malignancy and malignant revertants from which they were derived. Malignancy of these cells was associated with the ability to form colonies in agar. Cells of a line transformed by methylcholanthrene were malignant, formed almost no colonies in agar and the only chromosome change from the normal diploid chromosome banding complement was the addition of a long arm of chromosome 3. The results indicate that chromosome 3 carriers gene(s) that control malignancy in Chinese hamster cells in cell lines transformed by a viral or a chemical carcinogen and that malignancy was induced in both cell types by an increase of these genes.     

Cytogenetic effects of 2-methoxyethanol and its metabolite,methoxyacetaldehyde, in mammalian cells in vitro

Glycol ethers such as 2-methoxyethanol (2-ME) are reproductive toxins. The genotoxicity of 2-ME, especially its metabolites: methoxyacetaldehyde (MALD) and methoxyacetic acid (MAA), is not adequately investigated yet. We have shown previously that MALD induced mutation in the bacterial gpt gene which is inserted in an autosome of CHO-AS52 cell line but not in the hprt gene on the X chromosome of CHO-K1-BH4 cell line. These data suggest that MALD induces major deletion-type mutation. If this prediction is correct we would expect to observe that MALD is an efficient inducer of chromosome aberrations in both CHO cell lines. We have conducted a cytogenetic study using both CHO cell lines and human lymphocytes to investigate this phenomenon. Our results show that human lymphocytes treated with 10–30 mM MALD for 1 h or 0.05–0.5 mM MALD for 24 h induced significant dose-dependent increase of sister-chromatid exchanges (SCE) (p < 0.05). It also induced significant dose-dependent increase (p < 0.05) of chromosome aberrations in human lymphocytes (10–40 mM treated for 1 h, or 0.05–2.5 mM for 24 h) and in both CHO cell lines (1.25–20 mM for 3 h). Treatment of these cells with the parent compound, 2-ME did not induce chromosome aberrations nor SCE unless very high doses of the chemical were used. In conclusion, these results indicate that MALD is clastogenic to different cell types therefore it is potentially carcinogenic. The genotoxic effects of 2-ME in humans will be dependent upon the metabolic capability of individuals to bioactivate 2-ME to MALD.     

Concurrent detection of gene mutations and chromosome aberrations induced by five chemicals in a CHL/IU cell line incorporating a gpt shuttle vector

We previously established a transgenic Chinese hamster CHL/IU cell line, designated as KN63, for concurrent analysis of gene mutations and chromosome aberrations. The KN63 cell line contains copies of a shuttle vector with the Escherichia coli gpt gene as a mutational target in its chromosome. To evaluate the sensitivity of the cell line to various types of mutagens, methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), mitomycin C (MMC), vincristine sulfate (VIN) and C.I. basic red 9 hydrochloride (CIB) were assayed. KN63 cells were treated with each test chemical and gene mutations were detected in the gpt gene of the shuttle vector rescued from the KN63 cell genome into an E. coli host. Chromosome aberrations were concurrently evaluated by conventional metaphase analysis. MMS, ENU and MMC induced both gene mutations and structural chromosome aberrations in KN63 cells, with more efficient induction of the latter. VIN, a well-known aneugen, produced only numerical changes to chromosomes, while CIB was negative for both types of alteration. KN63 cells were as sensitive to MMS, ENU, MMC and VIN as Chinese hamster cell lines such as CHL, CHO and V79 cells. The characteristics of test chemicals indicated by this system should be useful for understanding endpoints in chemical mutagenesis.     

Differential sensitivity to x-ray of chromosomes of blood T-lymphocytes and B-and T-cell lines.

Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a singificantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia.     

Differential sensitivity to X-ray of chromosomes of blood T-lymphocytes and B- and T-cell lines

Summary Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a significantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia. Supported in part by USPHS grants CA-14413 and CA-16935.     

Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.     

SV40-transformed human fibroblasts exhibit a characteristic chromosomal pattern

A karyotype study of seven SV40-transformed human fibroblast cell lines was performed using R-banding. Although large variations existed from line to line and, to a lesser degree, from cell to cell in a given line, many common features were found. The most characteristic were chromosome imbalances. Some of the chromosomes or chromosome segments present in excess were, in decreasing order of frequency, the early replicating X, 12q, 3q, 12p, 19, 1p, and 6p. Losses of other chromosomes and chromosome segments were also frequent and involved 11p, 2p, 6q, 10p, 18, 4q, 8p, 4p, 10q, and 16. These imbalances seemed to correlate with metabolic characteristics, previously described, such as low activities of catalase (11p), superoxide dismutase 2 (6q), and acid phosphatase (2p, 11p) and a high ratio of lactate dehydrogenase B (LDHB, 12p) activity to LDHA (11p) activity.     

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.     

Investigation of cell line specific responses to pH inhomogeneity and consequences for process design

With increasing bioreactor volumes, the mixing time of the reactor increases as well, which creates an inhomogeneous environment for the cells. This can result in impaired process performance in large‐scale production reactors. Particularly the addition of base through the reactor headspace can be problematic, since it creates an area, where cells are repeatedly exposed to an increased pH. The aim of this study is to simulate this large‐scale phenomenon at lab‐scale and investigate its impact. Two different cell lines were exposed to pH amplitudes of a maximal magnitude of 0.05 units (pH of 6.95). Both cell lines showed similar responses, like decreased viable cell counts, but unaffected lactate levels. However, cell line B showed an initially increased specific productivity in response to the introduced amplitudes, whereas cell line A showed a consistently lower specific productivity. Furthermore, the time point at which base addition is started influences the impact, which pH amplitudes have on process performance. When pH control was started earlier in the process, maximal viable cell counts decreased and the lactate metabolic shift was less pronounced. These results show that the potential negative impact of pH amplitudes can be minimized by strategic process design.     

Re-evaluation of HeLa, HeLa S3, and HEp-2 karyotypes

In contrast to earlier reports, this study on HeLa, HeLa S3, and HEp-2 revealed that karyotypes of each cell line are characterized by a consistent marker chromosome composition and a constant number of copies for both normal and marker chromosomes. Based on these chromosome fingerprints and an analysis of 50 metaphases, the modal karyotype of each cell line was defined. Each modal karyotype had the composite content of the previously reported karyotypes of the same cell line, and, generally, the former had the same or a higher number of copies per chromosome than the latter. This modal karyotype can be used as a standard to identify and further individualize both the cell line itself and a subline within that cell line. We have also found that many cells within each cell line have the same karyotype. Portions of numerical data are compiled in a chart format by which the extent of chromosome differences between cultures can readily be compared. Also discussed in brief are characteristic chromosome changes that may help distinguish clonally derived cell lines from lines derived by cross-contamination.     

A genetic assay for aneuploidy: quantitation of chromosome loss using a mouse/human monochromosomal hybrid cell line

A genetic assay is described in which a mouse/human hybrid cell line R3-5 containing a single human chromosome (a monochromosomal hybrid) is used to detect chemically induced aneuploidy. In this assay the frequency of chromosome loss determined by the cloning efficiency of the cells in a selection medium is used as an index for the potential of a chemical to induce aneuploidy. The hybrid cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) and contain human chromosome 2, marked with Ecogpt, an E. coli gene for xanthine guanine phosphoribosyltransferase. These cells with a genotype of hgprt-/Ecogpt+ can grow in medium containing mycophenolic acid and xanthine (MX medium) but not in medium containing 6-thioguanine (6-TG). The loss of the human chromosome from R3-5 cells as a result of chemical treatment produces cells with a genotype of hgprt-/Ecogpt- which are capable of growth in the medium containing 6-TG. Thus, the cloning efficiency of cells treated with a test chemical in 6-TG provides a method to determine the frequency of cells that have lost the human chromosome. Two chemicals, colcemid and nocodazole, previously known to induce aneuploidy in mammalian cells were used for a preliminary evaluation of this test system. Both of these compounds at concentrations ranging from 0.002 to 0.032 micrograms/ml showed a concentration-related positive response in this assay.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

Metabolic response to low-level toxicant exposure in a novel renal tubule epithelial cell system

Toxicity testing is vital to protect human health from exposure to toxic chemicals in the environment. Furthermore, combining novel cellular models with molecular profiling technologies, such as metabolomics can add new insight into the molecular basis of toxicity and provide a rich source of biomarkers that are urgently required in a 21st Century approach to toxicology. We have used an NMR-based metabolic profiling approach to characterise for the first time the metabolome of the RPTEC/TERT1 cell line, an immortalised non-tumour human renal epithelial cell line that recapitulates phenotypic characteristics that are absent in other in vitro renal cell models. RPTEC/TERT1 cells were cultured with either the dosing vehicle (DMSO) or with exposure to one of six compounds (nifedipine, potassium bromate, monuron, D-mannitol, ochratoxin A and sodium diclofenac), several of which are known to cause renal effects. Aqueous intracellular and culture media metabolites were profiled by (1)H NMR spectroscopy at 6, 24 and 72 hours of exposure to a low effect dose (IC(10)). We defined the metabolome of the RPTEC/TERT1 cell line and used a principal component analysis approach to derive a panel of key metabolites, which were altered by chemical exposure. By considering only major changes (±1.5 fold change from control) across this metabolite panel we were able to show specific alterations to cellular processes associated with chemical treatment. Our findings suggest that metabolic profiling of RPTEC/TERT1 cells can report on the effect of chemical exposure on multiple cellular pathways at low-level exposure, producing different response profiles for the different compounds tested with a greater number of major metabolic effects observed in the toxin treated cells. Importantly, compounds with established links to chronic renal toxicity produced more diverse and severe perturbations to the cellular metabolome than non-toxic compounds in this model. As these changes can be rationalised with the different pharmacological and toxicity profiles of the chemicals it is suggested that metabolic profiling in the RPTEC/TERT1 model would be useful in investigating the mechanism of action of toxins at a low dose.     

Use of a human X mouse hybrid cell line to detect aneuploidy induced by environmental chemicals

A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome segregation following chemical treatment. The human chromosome present in the mouse cells can be readily identified by differential staining procedures. The frequency of cells containing 0 or 2 human chromosomes in the progeny of chemically treated monochromosomal hybrid cells provided a direct measure of aneuploidy. We tested the sensitivity of the proposed system with 3 model chemicals (colcemid, cyclophosphamide and benomyl) known to induce numerical or structural changes in chromosomes. The frequency of an abnormal segregation of the human chromosome was found to be dose dependent and consistently higher than controls. This system has the capability to detect gain as well as loss of a chromosome resulting from nondisjunction or other mechanisms leading to aneuploidy.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

Transforming DNA integrates into the host chromosome

A series of rat liver cotransformed cell lines have been constructed containing from 5 to 100 copies of a variant human growth hormone gene. We have used hybridization in situ to demonstrate that most, if not all, cotransformed sequences reside in a chromosome of the host cell. In each of four cell lines examined, hybridization was restricted to a single chromosomal site with no extrachromosomal sites apparent. The site was invariant within each line; however, each line revealed a different site of integration for transforming sequences. In two of the four lines, transforming DNA resided at or near the site of gross chromosomal rearrangements, in one line near an rDNA site, and in one line in the middle of an apparently normal chromosome. Thus, insertion is not restricted to a unique chromosome or chromosomal region.     

Phenylurea herbicides induce cytogenetic effects in Chinese hamster cell lines

The intensive use of herbicides over the last few decades has caused a general increase of environmental pollution. It is thus very important to evaluate the possible genotoxic properties of these chemical compounds as well as identifying their mode of action. Phenylurea herbicides are selective agents widely used for the control of infestant plants. Of these herbicides, which are widely used in agriculture, we analysed four of the less intensively studied molecules. More precisely, we investigated the genotoxic effects of fenuron, chlorotoluron, diuron, and difenoxuron by analyses of chromosomal aberrations (CAs) and sister chromatid exchange (SCE) in exposed mammalian cells. We used the Chinese hamster ovary (CHO) and epithelial liver (CHEL) cell lines, endowed with the absence or the presence, respectively, of an enzymatic system to activate pro-mutagenic compounds. Our results show that all herbicides tested induce, at high concentrations, an increasing number of CAs in non-metabolising CHO cells. Instead, in the exposed CHEL cell line, the four herbicides induced CAs also at the lowest dose-level. In the CHEL cells, a statistically significant increase of SCE was also observed. The phenylurea herbicides showed direct genotoxic activity, but the cytogenetic effects were greatly enhanced after metabolic conversion. These data, together with other information on phenylurea herbicides, are of great interest from the environmental point of view, and for human health. In fact, intensive use of herbicides contaminates soil, surface water, groundwater and agricultural products, and thus should be taken in particular consideration not only for those initiatives to specifically protect exposed workers, but also to safeguard the health of consumers of agricultural products.     

The influence of a substrate including extracellular matrix proteins on karyotypic variability in two cell lines of Indian muntjac skin fibroblasts

The influence of cell-culture conditions on numerical and structural karyotypic variability has been investigated in two Indian muntjac skin fibroblast “markerless” cell lines, M and MT. The cells were cultivated on a substrate consisting of extracellular matrix proteins (ECMs) synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for the chromosome number of the cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on a hydrophilic surface without ECM coating. These changes involve a significant decrease in frequency of cells with the modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. The MT cell line, differing from M line in the number of homologous chromosomes, exhibited a character of cell distribution similar to that of the M line for the chromosome number for only 1 day after cultivation on the ECM substrate, but not after 4 days under the same culture conditions, when no difference from the control cells was observed. Further cultivation of MT cells for 8 days did not change the character of cell distribution for the chromosome number relative to the control variant. The observed alterations seem to be due to disturbances in the correct chromosome-segregation process, which were caused by an abrupt shift in the cell-culture conditions. Analysis of the structural karyotypic variability revealed a significant increase in frequency of chromosomal aberrations in the M cell line for 1 and 4 days in culture on the ECM substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured under the same conditions. It can be suggested that the differing by the karyotypic structure, but the genetically identical cell lines have different response to the substrate. In contrast to the M line, in the MT line, a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate an MT cell on the given protein substrate while maintaining a balanced karyotypic structure characteristic of MT cell line.     

A new cell line of rat gasserian ganglion neurinoma NGUK-1

A new cell line has been derived from the rat gasserian ganglion neurinoma NGUK-1 induced by transplacental administration of ethylnitrosourea. It is characterized by an astrocyte-like growth pattern at the low cell density, and by an epitheliocyte-like pattern in the confluent monolayer. The cell line displays a high proliferative activity, its maximum mitotic index and proliferative pool being--2.5 and 96%, respectively. The chromosome number ranges between 20 to 100. The chromosome modal number is 39--44. The new cell line has been used for the express diagnostic of rabies, for determination of serum glial toxicity in neurologic patients, and interferon titration.     

Germ line specific factors in chemical mutagenesis

Chemical mutagenesis test results have not revealed evidence of germ line specific mutagens. However, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend upon the particular cell stage exposed. Many factors inherent in the germ line can be speculated to influence chemical transport to, and interaction with, target cell populations to result in mutagenic outcomes. The level of uncertainty regarding the general operation of such factors, in combination with the limited availability of chemical test data designed to address comparative somatic and germ cell mutagenesis, leaves open the question of whether there are mutagens specifically affecting germ cells. This argues for a conservative approach to interpreting germ cell risk from somatic cell mutation analysis.