Recovery of thioguanine-resistant cells from Chinese hamster V79 spheroids

Multicell spheroids may prove useful in evaluting the interactions of mutagens with cells exposed in a tissue-like environment. However, direct comparisons among populations of Chinese hamster V79 spheroids of different sizes or with monolayers are complicated by the observation that as spheroids enlarge, the fraction of mutant cells resistant to 6-thioguanine (TG^r) gradually decreases from about 5 in 10⁵ to less than 1 in 10⁵. There appear to be at least 2 explanations for these observations. First, TG^r cells grow less well as spheroids than do 6-thioguanine-sensitive (TG^s) cells. Second, the clonal nature of spheroid growth means that small samples fo spheroids are likely to contain fewer pre-existing TG^r cells.     

Antipain and 12-O-tetradecanoyl-phorbol-13-acetate: Phenomenology of effects on UV mutagenesis in V79 Chinese hamster cells

Antipain (AP) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were tested in V79 Chinese hamster cells for cytotoxicity and effects on survival and 6-thioguanine-resistant (6TG^r) mutation after UV-irradiation. AP and/or TPA were relatively non-cytotoxic and had no significant effects on UV survival. Despite their non-mutagenicity, the recovery of UV-induced 6TG^r colonies was significantly enhanced by the pretreatment with either AP (0.5–2 mM) or TPA (0.1–1 μg/ml) only during the expression period before the 6TG selection at a low density of cells in the absence of AP or TPA. Such enhancing effects were maximal when AP or TPA was present during the late expression period after the mutation fixation and extensive dilution of DNA lesions. Reconstruction experiments revealed the antagonistic actions that TPA and AP tended to eliminate and increase, respectively, the metabolic co-operation. In the TPA-plus-AP treatment, AP abolished the TPA-enhanced recovery of induced mutants. Thus, it seems that TPA increases the mutant recovery largely through decreased metabolic co-operation and AP could modulate the mutation expression. Further, an error-prone inducible repair may not exist or, if it exists, AP may not inhibit it in V79 Chinese hamster cells.     

Effects of a tumor promoter and an anti-promoter on spontaneous and UV-induced 6-thioguanine-resistant mutations and sister-chromatid exchanges in V79 Chinese hamster cells

The effects of a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or an anti-promotor antipain (protease inhibitor) on spontaneous and ultraviolet-induced sister-chromatid exchanges (SCEs) and 6-thioguanine- resistant (6TG^r) recessive mutations were examined in V79 Chinese hamster cells in culture. TPA and/or antipain neither significantly altered base-line and UV-induced immediate SCE frequencies, nor decreased the level of delayed SCEs which persisted 6–7 days after irradiation. TPA and/or antipain appeared to enhance the recovery of UV-induced 6TG^r colonies at the plateau expression phase despite non-mutagenicity by themselves and unaltered metabolic co- operation. Thus, the results conceivably imply that the 6TG^r-recessive mutation expression, but not fixation, can be modulated at the cell level by the TPA and/or antipain. Our results, together with the recent results of Loveday and Latt, may argue against the notion that TPA enhances the antipain-suppressible SCEs as an index of mitotic recombination in relevance with a tumor-promotion mechanism.     

Effects of phorbol myristate acetate,phorbol dibutyrate,ethanol, dimethylsulfoxide,phenol, and seven metabolities of phenol on metabolic cooperation between chinese hamster v79 lung fibroblasts

The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS Chemical Abstracts Service - DMSO dimethylsulfoxide - ETOH ethanol - HGPRT hypoxanthine-guanine phosphoribosyl transferase - HGPRT+ HGPRT-competent - HGPRT– HGPRT-te]deficient - MC metabolic cooperation - MC+ metabolic cooperation-competent - MC– metabolic cooperation-deficient - MEM minimum essential medium - PDBu phorbol dibutyrate - PMA phorbol myristate acetate - 6TG 6-thioguanine - 6TG^r 6-thioguanine-resistant - 6TG^s 6-thioguanine-sensitive - V79/MC assay Chinese hamster V79 lung fibroblast assay for metabolic cooperation     

An enumerative assay of purine analogue resistant lymphocytes in women heterozygous for the Lesch-Nyhan mutation

In females heterozygous for the Lesch-Nyhan (LN) mutation, there is mosaicism of peripheral blood lymphocytes (PBLs) with regard to sensitivity to 6-thioguanine (TG) inhibition of tritiated thymidine ([³H]Tdr) incorporation following phytohemagglutinin (PHA) stimulation. That there are two populations of PBLs, normal and mutant (LN-like), has been demonstrated by an autoradiographic enumerative assay. A single three-generation family containing six potentially heterozygous females was studied. Five of the six were mosaics with frequencies of TG-resistant (TG^r) PBLs ranging from 1.4×10^–3 to 4.2×10^–3 when tested at 2×10^–4 m TG. The median frequency of TG^r PBLs in 63 healthy non-LN individuals between the ages of 11 and 75 years was found to be 1.1×10^–4 (mean 1.3×10^–4) (10th and 90th percentiles—6.1×10^–5 and 2.1×10^–4) and was not age related. The sixth potentially heterozygous female in the current family had a TG^r PBL frequency of 1.9×10^–4. In the five females with elevated TG^r PBL frequencies, TG^r skin fibroblasts with frequencies ranging from 26% to 100% of the sample tested were found; in the female with the normal TG^r PBL frequency, no TG^r skin fibroblasts were found. The former group was considered to be LN heterozygous. Four of the five had been previously diagnosed as such. The latter individual is considered to be genotypically normal. Females who are heterozygotes for the LN mutation have two populations of PBLs.This work was supported by National Institutes of Health Grant No. PHS RO1 15450 and the Surgical Associates Foundation, Inc., University of Vermont College of Medicine. Elizabeth F. Allen was supported by the NIH National Research Service Award No. 5 F32 CA 05918.     

Spontaneous 6-thioguanine-resistant lymphocytes in Fanconi anemia patients and their heterozygous parents

Summary The incidence of spontaneous 6-thioguanine-resistant (TG^r) lymphocytes was studied in the peripheral blood collected from seven Fanconi anemia (FA) patients and five of their heterozygous parents using an autoradiographic or a lymphocyte cloning method. Five of the seven patients showed a significantly elevated incidence of TG^r lymphocytes as compared to age- and sex-matched healthy controls. There was, however, no difference between FA heterozygotes and controls. These results suggest some variability among the patients similar to those reported in clinical and cytogenetic investigations. The basis for the increase in TG^r cells in the patients is not known, but the inherent genomic instability reflected as increased frequencies of chromosomal aberrations is one possible explanation.     

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum

Summary To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a supersuppressor selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HAT^r) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT mutants — selected by their resistance to 6-thioguanine (TG^r) — and TK^- mutants — selected by their resistance to 5-bromodeoxyuridine (BrdUrd^r) — are HAT^s. Therefore, from HPRT^- TK^- double nonsense mutants, whose phenotype would be TG^r BrdUrd^r (HAT^s), simultaneous HPRT⁺ TK⁺ double phenotypic revertants could be obtained by selecting HAT^r (TG^s BrdUrd^s) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TG^r variants, 53 of which were HAT^s and the rest HAT^r. Among 36 TG^r (HAT^s) variants tested, 23 did not revert to HAT^r, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT^- nonsense mutants because they were very stringent (had less than 2% of wild-type [³H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrd^r) in 7 of these strains allowed us to isolate 29 TG^r BrdUrd^r (HAT^s) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HAT^r (TG^s BrdUrd^s) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.     

A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation.

A mammalian cellular system, utilizing Syrian hamster embryo cells, was developed for the concomitant study of neoplastic transformation and somatic mutation. Chemically induced somatic mutation of the cells was assayed at two genetic loci. Mutants deficient in hypoxanthine phosphoribosyl transferase (HPRT) were detected by the production of colonies resistant to 8-azaguanine (AG^r) or 6-thioguanine (TG^r) and mutants with an altered Na⁺/K⁺ ATPase were detected by the production of colonies resistant to ouabain (Oua^r). Colonies resistant to each of the three selective agents were isolated and characterized. AG^r and TG^r resistant cells maintained their resistance to the selective agent after isolation and growth in the absence of the drug, displayed a low reversion frequency, and possessed less than 1% of the HPRT activity of the wild-type cells. AG^r cells were also resistant to the cytotoxicity effects of 6TG. Oua^r cells also maintained their resistance to ouabain and were less sensitive to the inhibition of ⁸⁶Rb uptake by ouabain than the wild-type cells. The spontaneous frequency of all three types of resistant cells was <10^?6, but the mutation frequency was significantly increased following exposure of the cells to known mutagens in a dosage-dependent manner. These properties indicate that AG^r and TG^r cells posess a mutation in the structural or regulatory gene for HPRT, and that Oua^r cells have an altered Na⁺/K⁺ ATPase.The factors involved in the quantification of the mutation frequencies of hamster embryo cells following exposure to carcinogens were determined. Cytotoxicity was assayed by a reduction in the cloning efficiency of the treated cells. The recovery efficiencies of the resistant cells were measured by reconstitution experiments and the degree of cross feeding effects of HPRT^? cells was determined. The expression time of the mutations following exposure of the cells to carcinogens was also examined, and the mutation frequencie at the two loci of hamster embryo cells following exposure to MNNG or benzo(a)-pyrene (B(a)P) were determined. Employing this system, a quantitative comparison can be made between the frequencies of somatic mutation and morphological transformation.     

Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 chinese hamster cells,independence of cytotoxic and mutagenic responses

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TG^RHGPRT^? mutants but were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70 % survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU ENU EMS MMS ? DMS. MNU was approx. 20 times more effective than MMS and DMS.Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O⁶/N⁷ ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10⁵ cells/μM O⁶-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.     

Isolation and characterization of amphotericin B-resistant cell lines in Chinese hamster cells

Amphotericin B is a polyene macrolide antibiotic which interacts specifically with steroids in mammalian cell membranes. Amphotericin B-resistant (AMB^r) lines of stable phenotype have been isolated from cultured Chinese hamster (V79) cells. Three AMB^r clones (AMB^r-1, -2 and -3) isolated independently after treatment with nitrosoguanidine were resistant to ≥150 μg/ml of the antibiotic, while DNA synthesis as well as the colony-forming ability of the parental V79 cells was blocked by >80% of control in the presence of 20–50 μg/ml amphotericin B. The AMB^r cell line also exhibited increased resistance to other polyene macrolide antibiotics such as nystatin and pentamycin. Other agents, however, such as cytosine arabinoside or ricin, blocked DNA synthesis in AMB^r cells to the same extent as in V79 cells. The amphotericin B resistance phenotype was stably retained even after AMB^r cells were cultured in the absence of the drug for over 200 generations. The content of free cholesterol or its esters was significantly decreased in all three resistant clones. Furthermore, cholesterol synthesis from acetate as well as mevalonate was partly defective in AMB^r cells, compared with that in V79 cells.     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. II. The effect of posttreatment with non-toxic concentrations of thymidine

The ability of posttreatment exposure to non-toxic concentrations of thymidine (TdR) to enhance the lethal effects of a number of alkylating agents, X-rays and UV and the lethal and mutagenic effects of N′-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) has been examined in V79 cell lines. TdR posttreatment enhanced the cytotoxic effects of ethyl methanesulphonate (EMS), MNU and ENU but not of UV or X-rays and increased both the spontaneous and MNU- and ENU-induced frequencies of azaguanine resistant (AZ^R) mutants. No significant effect of TdR on the spontaneous frequency of thioguanine resistant (TG^R) mutants was demonstrated but the frequency of MNU-induced mutants to TG^R was enhanced. The effects on expression of both potentially lethal and premutagenic damage were reversed by addition of an equimolar concentration of deoxycytidine (dCdR). The enhancement in spontaneous and induced mutant frequency (IMF) at the HGPRT locus appears to be due to an alteration in the selective efficiency of purine analogous due to alteration in growth kinetics of cells exposed to TdR or treated with alkylated agents or posttreated with thymidine after alkylation damage and not to an alteration in the miscoding potential of alkylated bases.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

The cytotoxic and mutagenic effects of 5-bromodeoxyuridine-plus-black-light treatment in cultured mammalian cells

The cytotoxic and mutagenic effects of the incorporation of 5-bromodeoxyuridine (BrdU)_followed by exposure to black light were investigated with Chinese hamster ovary (CHO) cells in cell culture. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus was determined by selection for 6-thioguanine resistant (TG^r) mutants (CHO/HGPRT system). BrdU alone has been shown to be mutagenic only at concentrations of 50 μM or greater. This study was performed in an effort to determine whether BrdU is actually incorporated into the hgprt gene when lower, nonmutagenic concentrations are employed. Neither BrdU (1–20 μM) nor exposure to black light alone was mutagenic, but the combined treatment did result in the induction of TG^r mutants. The mutant frequency increased with increasing light exposure at constant BrdU and with inreasing BrdU at constant light exposure. These results show that BrdU is incorporated into the hgprt gene, but that this does not result in mutation induction in the absence of light exposure. Such a BrdU-plus-light procedure might be applied to studies of DNA repair at this locus, since mutation induction requires both BrdU incorporation and subsequent exposure to black light.     

Evidence for electrogenic bicarbonate transport in Amphiuma small intestine

Isolated segments of small intestine of Amphiuma were short-circuited in buffer containing bicarbonate. Theophylline (10 mM) increased short circuit current (I_sc) in proportion to the bicarbonate concentration in the bath. The theophylline-stimulated I_sc was rapidly reduced, though not abolished, in the presence of acetazolamide at concentrations as low as 10^?6 M. Unidirectional fluxes of ²²Na and ²⁴Na in paired intestinal segments in Cl-free buffer reveal that the increase in I_sc produced by theophylline is not accounted for by an increase in net sodium flux. These results suggest that theophylline stimulates an electrogenic secretion of bicarbonate.     

Evidence for functional hemizygosity at the Emtr locus in CHO cells through segregation analysis

The hypothesis of functional hemizygosity at the emetine-resistant (Emt^r, a non-X-linked recessive marker) locus in Chinese hamster ovary (CHO) cells has been examined by segregation analysis. The frequencies and the rates of segregation of the Emt^r and Thg^r (thioguanine-resistant, an X-linked recessive mutation) markers were determined from hybrids constructed between an Emt^r-Thg^r CHO cell line and various other Chinese hamster lines (V79, M3-1, CHO, GM7S, CHW and CHL). Thg^r segregants were obtained at similar frequencies (10^?2–10^?3) from all the hybrids. The frequency of segregation of the Emt^r marker, however, was similar to that of Thg^r only in the CHO × CHO hybrids and was much lower (10^?4–10^?6) than the CHO × other Chinese hamster hybrids. Similar results were obtained when the segregation rates for the two markers from various hybrids were determined. These results are consistent with the hypothesis that in CHO cells, the gene responsible for Emt^r is present in a single (functional) copy, whereas two copies of this gene are present in other Chinese hamster lines examined.     

N6,O2′-dibutyryl adenosine 3′,5′-monophosphate-stimulated release of proteoglycans from cultured immature rabbit ear cartilage

The stimulatory effects of N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases.     

In vitro induction,expression and selection of thioguanine-resistant mutants with human T-lymphocytes

Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G₀ phase peripheral blood mononuclear cells from a single individual. After explosure to γ-irradiation from a ¹³⁷Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TG^r) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 × 10⁴ cells/well. The development of this in vitro assay should allow direct study of susceptibility to γ-irradiation in the human population in terms of both cytotoxicity and mutation induction.     

Synergistic inhibition of metabolic cooperation by oleic acid or 12-0-tetradecanoylphorbol-13-acetate and dichlorodiphenyltrichlorethane (DDT) in Chinese hamster V79 cells: Implication of a role for protein kinase C in the regulation of gap junctional intercellular communication

The effects of TPA and/or DDT and oleic acid and/or DDT on gap junction-mediated intercellular communication (i.e. metabolic cooperation) between Chinese hamster V79 cells was examined. Addition of TPA, DDT or oleic acid alone to cocultures of 6t-hioguanine-resistant (6-TG ^R ) and 6-thioguanine-sensitive (6-TG ^S ) V79 cells significantly increased the recovery of 6-TG ^R cells indicating inhibition of metabolic cooperation. In the presence of TPA and DDT or oleic acid and DDT the observed recovery of 6-TG ^R cells was significantly greater than the expected (calculated) additive 6-TG ^R cell recovery. No synergistic increases in 6-TG ^R cell recovery were observed when co-cultures of V79 cells were exposed to dieldrin and DDT. These results indicate that TPA and DDT or oleic acid and DDT can act synergistically to inhibit metabolic cooperation. These data suggest a role for protein kinase C in the regulation of gap junction-mediated intercellular communication.Abbreviations DDT dichlorodiphenyltrichlorethane - MC metabolic cooperation defective - 6-TG 6thioguanine - TPA 12-0-tetradecanoylphorbol-13-acetate     

Stromal Adipocyte Enhancer-binding Protein (AEBP1) Promotes Mammary Epithelial Cell Hyperplasia via Proinflammatory and Hedgehog Signaling

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1^TG) mice, and the onset of ductal hyperplasia was accelerated in AEBP1^TG mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1^TG bone marrow cells into non-transgenic (AEBP1^NT) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1^TG mammary macrophages and epithelium. Shh expression was induced in AEBP1^TG macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1^TG mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1^TG peritoneal macrophages. The conditioned AEBP1^TG macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1^TG macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.     

Use of the cytokinesis-block method for the analysis of munuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze munuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for munucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 μg/ml CYB for varying durations (8–48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for munucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of munuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125–1.0 μg/ml) and cyclophosphamide (2–12 μg/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in munucleus frequency over controls at the highest concentration tested (1.0 μg/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in munucleus frequency over controls at the highest tested concentration (12 μg/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze munucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.