Morphological transformation of BALB/3T3 cells by various procarcinogens in the presence of a rat liver S-9 activation system

An Aroclor-induced rat hepatic S-9 metabolic activation system was incorporated into the BALB/3T3 cell transformation assay to increase its sensitivity to a wide range of procarcinogens. S-9 was prepared from Aroclor 1254-induced (500 mg/kg) Fischer 344 rats. Cyclophosphamide, dimethylnitrosamine, 2-aminofluorene, and 2-naphthylamine were metabolized to reactive forms capable of inducing both dose-dependent toxicity and morphological transformation of BALB/3T3 cells. Treatments without an exogenous metabolic activation system were nontoxic and nontransforming. Adaptation of this commonly used exogenous metabolic activation system to BALB/3T3 cells will allow detection of the transforming potential of procarcinogens which test negative in a standard assay.     

两种检测多环芳烃致癌性细胞转化实验方法的研究

目的 比较BALB/c-E6E7细胞(含HPV16 E6E7基因的BALB/c 3T3细胞)转化实验与BALB/c 3T3细胞转化实验对三种多环芳烃致癌性的评价能力.方法 应用已优化的BALB/c-E6E7细胞转化实验与Ⅱ阶段BALB/c3T3细胞转化实验检测三种多环芳烃的致癌性和促癌性.结果 两种细胞转化实验对三种多环芳烃致癌性和促癌性的评价结果一致,且符合IARC对其致癌性的分类标准.BALB/c-E6E7细胞可形成更多的转化灶,转化频率约为BALB/c 3T3细胞的2～10倍,该细胞转化实验具有更高的敏感性并可有效缩短实验时间.结论 BALB-E6E7细胞转化实验是一种更为敏感和高效的评价多环芳烃致癌性和促癌性的方法,有助于环境致癌物的风险评估.     

Detection of DNA strand breaks, DNA-protein crosslinks, and telomerase activity in nickel-transformed BALB/c-3T3 cells.

Although nickel compounds are known carcinogens, the underlying carcinogenic mechanisms are not fully understood. The objective of this research was to determine if the genotoxic lesions of DNA strand breaks and DNA-protein crosslinks are present in nickel-transformed BALB/c-3T3 cells, and to further elucidate the potential carcinogenesis of insoluble and soluble nickel compounds through telomerase activity in nickel-transformed BALB/c-3T3 cell lines. DNA strand breaks, DNA-protein crosslinks and telomerase activity were investigated by single cell gel electrophoresis (comet assay), (125)I-postlabelling techniques, and the TRAP-silver staining assay, respectively. Results showed that both DNA strand breaks and DNA-protein crosslinks were present in nickel-transformed BALB/c-3T3 cells. However, the highest levels of DNA strand breaks and DNA-protein crosslinks were found in insoluble crystalline NiS-transformed cells and high levels of DNA strand breaks and DNA-protein crosslinks were also found in the transformed cells induced by two water-soluble NiCl(2) and NiSO(4) at moderate concentrations of cytotoxicity. These data suggest that these two genetic endpoints are useful biomarkers and are associated with cell transformation and carcinogensis of insoluble and soluble nickel compounds. Also, we found that the crystalline NiS- and NiCl(2)-transformed cells possessed a high telomerase activity. A weak telomerase was found in NiSO(4)-transformed cells. The results seem to indicate that in addition to crystalline NiS, some water-soluble nickel compounds such as NiCl(2) are also highly carcinogenic. These results may partly explain the cell transformation and relative carcinogenic potency of insoluble crystalline NiS, soluble NiCl(2), and NiSO(4).     

In vitro cytotoxic and cell transforming activities exerted by the pesticides cyanazine,dithianon, diflubenzuron,procymidone, and vinclozolin on BALB/c 3T3 cells

Cytotoxic and cell transforming activities of the pesticides cyanazine, diflubenzuron, dithianon, procymidone, and vinclozolin were investigated in vitro by utilizing the BALB/c 3T3 cell transformation test performed in the presence or in the absence of S-9 mix as an exogenous bioactivation system for the chemicals. All the assayed pesticides were cytotoxic in the absence of S-9 mix, whereas only dithianon exerted cytotoxic effects in the presence of metabolic activation. All the chemicals tested did induce BALB/c 3T3 cell transformation, to a various extent, in the absence of S-9 mix. Cell transforming ability of cyanazine and diflubenzuron was not detectable in the presence of S-9. © 1993 Wiley-Liss, Inc.     

Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens.

BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B1 [AFB1], and 4-nitroquinoline-N-oxide [4-NQO]). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUAR) was examined, we found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUAR locus in this system, while AFB1 was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (ClN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. ClN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.     

BALB/c 3T3细胞转化实验的优化及其在致癌物协同作用研究中的应用

目的 优化BALB/c 3T3细胞转化实验,并应用于致癌物间协同致癌作用的研究.方法 从血清浓度、培养基类型和致癌物作用时间三个因素对BALB/c 3T3细胞转化实验进行优化.采用优化的实验方案,选择致癌物作用7 d后重新接种,再以含5%胎牛血清的DMEM/F12(1:1)培养基培养进行细胞转化实验,对致癌物间的协同作用进行检测,通过小鼠体内致瘤实验对转化灶细胞的恶性特征进行验证.结果 二乙基亚硝胺(DEN)与2、3、7、8-四氯二苯并二恶英间有较强的协同致癌作用.微囊藻毒素单独具有较强促细胞恶性转化能力,但这种促转化能力却受到DEN的抑制.实验诱发的转化灶具有Ⅱ型转化灶的特征,并可在体液与细胞免疫缺陷(SCID)小,鼠体内致瘤.结论 经优化的BALB/c 3T3细胞转化方案既充分模拟了致癌物联合作用的方式,又缩短了实验周期,可有效应用于致癌物间协同作用的研究.     

Detection of non-genotoxic carcinogens in the BALB/c 3T3 cell transformation/mutation assay system

We have employed BALB/c 3T3 cells in a simultaneous cell transformationand mutation assay protocol to see whether both genotoxic andnon-genotoxic carcinogens can be identified. Three known mutagenicanimal carcinogens tested positive for transforming activity:dimethylnitrosamine, methylcholanthrene and methylnitrosourea.In addition, we tested three non-mutagenic compounds-two humancarcinogens, benzene and diethylstilbestrol, and the possiblehuman carcinogen dichlorodiphenyltrichloroethane; all threedisplayed transforming activity. These results add to existingdata supporting the validity of the BALB/c 3T3 cell transformationsystem as a reliable short-term in vitro test for carcinogensin general, and for non-genotoxic carcinogens in particular. ¹To whom correspondence should be addressed     

An age-related γδ T cell suppressor activity correlates with the outcome of autoimmunity in experimental Trypanosoma cruzi infection

In this work the suppressive activity of splenic T cells from young and aged BALB/c mice infected with Trypanosoma cruzi were compared and correlated with the development of autoimmune myocarditis. The T cells from young adult BALB/c mice with acute T. cruzi infection exhibit suppressor activity when added to full allogeneic or Mis-disparate mixed lymphocyte cultures. This suppression could not be reverted by exogenous interleukin (IL)-2 and was not directly dependent on the presence of IL-4, IL-10 or transforming growth factor-β. Further characterization of the T cell lineage responsible for the suppressor activity by in vitro and/or in vivo depletion with monoclonal antibody to αβ or γδ T cell receptor revealed that splenic γδ T cells function as suppressor lymphocytes in young T. cruzi-infected mice. In addition, these young adult BALB/c mice do not develop autoimmune myocarditis and showed a low incidence of syngeneic heart graft rejection in the early chronic phase of the infection. In contrast, T cells from acutely infected aged BALB/c mice lacked demonstrable T suppressor activity. Furthermore, these mice developed a severe autoimmune myocarditis as early as 2 months after the onset of the infection, when the majority of them reject syngeneic heart grafts. These findings suggest that a γδ T cell-mediated suppressor mechanism may operate in the avoidance of the breaking of tissue-specific tolerance during the acute infection. Moreover, such a mechanism is likely related to the immune system chronobiology.     

Methodology for cell transformation assays with C3H/10T1/2 mouse embryo fibroblasts

Summary Basic cell culture conditions required for execution of cell transformation studies with C3H/10T1/2 mouse embryo fibroblasts are described. These protocols permit assessment of the ability of chemicals to convert nontumorigenic C3H/10T1/2 cells to a tumorigenic phenotype characterized by altered controls upon in vitro cell division. Recent assay modifications that enhance the sensitivity of this focus formation assay and permit the study of multistage transformation processes are noted.     

In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane

The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 micrograms/ml, did not induce transformation, whereas dioxane was very active in the induction of type III foci in the cultured BALB/3T3 cells.     

Selection of fetal bovine serum for use in the C3H/10T1/2 CL8 cell transformation assay system

The purpose of this communication is to report our experience concerning the variation in cloning efficiency and transformation frequency utilizing C3H/10T1/2 CL8 cells with 23 different lots of fetal bovine sera. These sera were purchased from five different commercial sources. The standard cell transformation assay using 1,000 cells per dish and 3-methylcholanthrene (7.5 μg/ml) as the transforming agent was performed. The chemical exposure period was 3 days. The cloning efficiency was determined in parallel toxicity tests using 200 cells per dish. Only three out of 23 serum lots supported a strong response in cell transformation. The results indicated that variation in the ability of sera to support cell transformation was not supplier dependent. In addition, our results showed that serum lots exhibiting the best cloning efficiencies did not necessarily support cell transformation. It is apparent that reliance on cloning efficiency alone would be inadequate as a means of selecting a serum lot. We therefore recommend that a complete cell transformation assay be performed when selecting fetal bovine serum for use in this assay.     

Cell-cycle deregulation in BALB/c 3T3 cells transformed by 1,2-dibromoethane and folpet pesticides

The cell-transforming potential of 1,2-dibromoethane and folpet, two widely used agricultural pesticides that are potential sources of environmental pollution, has been previously ascribed to their promoting activity. In this study, we investigated whether BALB/c 3T3 transformation by these chemicals was associated with the deregulation of signals involved in cell-cycle progression and in cell-cycle checkpoint induction. We found that two BALB/c 3T3 cell clones transformed by in vitro medium-term (8-week) exposure to the carcinogens had a constitutive acceleration of cell transition from G(1) to S phase and an abrogation of the radiation-induced G(1)/S checkpoint. These events involved multiple signals; in particular, the inhibitors of cyclin/cyclin-dependent kinase complexes p21 and p27 were significantly down-modulated and the positive regulators of cell-cycle progression cyclin D(3) and E were up-modulated. As anticipated for cells where the G(1)/S checkpoint was abrogated, the transformed cells exhibited a significant reinforcement of the radiation-induced G(2)/M checkpoint, the only checkpoint remaining to protect genomic integrity. However, cyclin A(1) and B(1) coexpression and cyclin A(1) overexpression were found despite the G2 arrest in irradiated cells and these signals likely attenuate the G(2)/M checkpoint. These alterations to normal cell cycling may promote the emergence of both numerical and structural chromosomal abnormalities and their tolerance. Such a condition could play a key role in neoplastic transformation and be crucial in tumor progression. Furthermore, cyclin A(1) overexpression may play an autonomous role in the neoplastic transformation of BALB/c 3T3 cells, as it does in other cell types of mesenchymal origin.     

3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone [MX] shows initiating and promoting activities in a two-stage BALB/c 3T3 cell transformation assay

A transformation assay using BALB/c 3T3 cells was conducted on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to assess initiation and promotion activities of MX carcinogenesis. Statistically significant positive responses were obtained compared with the corresponding solvent controls in both the initiation assay post-treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) and the promotion assay pretreated with 3-methylcholanthrene (MCA). Both TPA and MX inhibited metabolic cooperation in an assay using co-culture of V79 6-thioguanine (6-TG) sensitive and insensitive cells. However, cells isolated from transformed foci in the initiation assay did not induce any nodules after inoculation to BALB/c mice, the strain of mouse from which the transformation assay cells were derived. Although the study was carried out for 2-3 weeks, this might have been too short to develop nodules under the conditions of this experiment. This in vitro cell transformation study with MX adds supportive information to studies showing MX carcinogenicity and tumour promoter activity, and adds mechanistic understanding of the action of MX.     

Crocidolite induces cell transformation and p53 gene mutation in BALB/c-3T3 cells

Cell transformation is one of the most common assays used to study morphological changes in the multistep process of carcinogenesis. The present study was initiated to investigate the ability of crocidolite to induce cell transformation in BALB/c-3T3 cells and to analyze the relationship between p53 mutations and crocidolite-induced cell transformation, if any. Cell transformation was carried out according to standard procedures. Exponentially growing cells were exposed to different concentrations (0.2-20 microg/cm(2)) of crocidolite fibers for 72 h. Foci obtained from cell transformation were analyzed for their ability to grow in soft agar (anchorage-independence) and p53 alterations. The results of this study demonstrate that there was an increase in transformation frequency (TF) with an increase in concentration of crocidolite. Also, focal cells were able to grow on soft agar, indicating anchorage-independence. cDNA was prepared from RNA isolated from Type 3 foci and subjected to mutational analysis. Eleven exons of the p53 gene from eight transformed cell lines were analyzed for alterations using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Alterations were found in seven of eight cell lines, two of them were in exons 4-6, and five in exons 9-11. The alterations were randomly scattered among the crocidolite dose groups. These results suggest that crocidolite induces mutations predominantly in exons 9-11 of the p53 gene in a nondose-dependent manner.     

Preneoplastic potential of morphologically distinct transformed foci induced by 3-methylcholanthrene

Individual variability of scoring foci positive for transformation presents a difficult problem in assessing the transformation assay. In this study, an attempt was made to identify five morphologically distinct types of transformed foci based on size (2–3, 3–4, and ≥4 mm in diameter), invasiveness (smooth vs. invading margins), and other properties (piling vs. spread) induced by 3-methylcholanthrene in Balb/c-3T3 cells. The transformed focal cells were used in in vitro studies including anchorage-independent analysis, focal reconstruction, gene transfection using NIH-3T3 host cells, and Southern blotting to assess amplification of five proto-oncogenes (K-ras, H-ras, c-fos, c-jun, c-myc) and a tumor suppressor (p53) gene. Results showed that 1) there was a significant increase in anchorage-independent growth of all five types of foci ranging from 7–12%; 2) all five morphological types of transformed foci showed 8–15% focal reconstruction; 3) DNA from all five types of transformed foci induced transformation in NIH-3T3 cells at a level significantly above the control DNA; 4) gene amplification studies indicated amplification in both K-ras and H-ras proto-oncogenes; however, c-fos, c-jun, and c-myc did not show DNA amplification. The tumor suppressor gene (p53) was activated and the increase was up to 3-fold over the normal Balb/c-3T3 DNA. These findings are consistent with our hypothesis that all five morphologically different foci have preneoplastic potential and that any foci of size ≥ 2 mm regardless of invasiveness and piling should be scored as positive during the transformation assay. Environ. Mol. Mutagen. 32:369–376, 1998 © 1998 Wiley-Liss, Inc.     

CD3AK细胞过继免疫治疗的实验研究

目的: 分析抗CD3分子的单克隆抗体 (mAb)yCD3的免疫学特性和生物学活性, 观察其激活的免疫活性细胞CD3AK体外及动物体内的抑瘤作用。方法: 用流式细胞术(FCM)测定yCD3的特异性, 以及CD3AK细胞的免疫表型和产生细胞因子的情况。用 3H -TdR法测定yCD3对淋巴细胞转化的作用; 乳酸脱氢酶法 (LDH)测定CD3AK细胞的体外细胞毒活性。建立荷瘤小鼠模型, 观察静脉注射CD3AK细胞后肿瘤生长的情况、转移灶数量和小鼠存活天数。结果: yCD3与T细胞呈特异性反应, 5μgyCD3可竞争抑制 70%的标准抗CD3抗体与细胞表面CD3分子的结合。yCD3刺激外周血淋巴细胞增殖的有效浓度为 8μg/L, 并与IL- 2、抗CD28抗体有协同作用。活化的CD3AK细胞中CD3 、CD8 和CD25 细胞增多; 产生IL- 2和IFN γ的CD3 细胞均有不同程度的增加, 在抗CD28抗体协同刺激下分别增加 3. 29和 2. 47倍。当效靶细胞比为 80∶1时, CD3AK细胞对体外肿瘤细胞杀伤的百分率为 57. 54%。分组观察荷瘤动物, CD3AK细胞治疗组的抑瘤率为 33. 17%, 对小鼠肿瘤肺转移的抑制率为39. 70%, 与LAK细胞联合治疗的疗效更显著。结论: yCD3可活化T细胞, 诱导的CD3AK细胞在体外及动物体内显示抑制肿瘤的作用, 在临床抗肿瘤过继性免疫治疗中具有重要意义。     

Transformation of 3T3 cells with Abelson virus proviral DNA

Abelson murine leukemia virus-integrated proviral DNA was found to induce oncogenic transformation in both NIH/3T3 and BALB/3T3 cell lines of murine fibroblasts with a rather low efficiency. The transformation occurs in absence of a replicating helper virus. The clones derived from foci of DNA-treated cells show characteristics of Abelson virus-transformed trals.     

Morphological transformation of C3H/10T1/2 CL8 cells by procarcinogens

In order to increase the sensitivity of the C3H/10T1/2 CL8 (10T1/2) cell transformation system, we increased the chemical exposure period to a total of 6 days (two consecutive 3-day exposures). Using this modified procedure, we transformed 10T1/2 cells with procarcinogens such as aflatoxin B1, benz(a)anthracene, and 4-nitroquinoline-1-oxide which have been negative in the standard 10T1/2 cell transformation assay. However, beta-naphthylamine was inconclusive and 2-acetylamino-fluorine was negative in this modified assay system. Our results demonstrate that a simple modification of the 10T1/2 cell transformation method can increase the sensitivity to some procarcinogens that require metabolic activation.     

Dose-response studies on neoplastic transformation of BALB/3T3 clone A31-1-1 cells by aflatoxin B1, benzidine, benzo[a]pyrene, 3-methylcholanthrene, and N-methyl-N'-nitro-N-nitrosoguanidine

The BALB/3T3 clone A31-1-1 mouse embryo cell line at passages 7 to 13 was selected for morphologic studies of neoplastic transformation by carcinogens of different chemical classes, in the absence of any added extracellular metabolic activation. Dose-related transforming activity was demonstrated for the carcinogens aflatoxin B1 (AFB) and benzidine (BZ) not previously reported in this system, and was confirmed for benzo[a]pyrene (BP), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Spontaneous transformation per cells at risk was low (0.14 type III foci x 10(-4), while chemically induced transformation was 2 to 3 orders of magnitude higher with all compounds. The molar concentration of carcinogens in complete medium, required to induce a transformation frequency of 1.0 type III foci x 10(-3) showed the highest level of activity for BP (0.04 microns), an intermediate level for AFB (0.2 to 1.4 microns), MCA (1.1 micron), and MNNG (2.3 microns), and the lowest level of activity for BZ (30.0 microns). The dose-related induction of morphological transformation in this clone by carcinogens of different classes indicates the potential value of this biological system in quantitative studies of carcinogen combinations, especially at low dose levels.     

Overexpression of autocrine motility factor receptor (AMFR) in NIH3T3 fibroblasts induces cell transformation

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of 78000 molecular weight (gp78), regulating cell motility signaling in vitro and metastasis in vivo. To test whether AMFR could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 fibroblast cells with expression vectors carrying the full-length cDNA for mouse AMFR and evaluated the effects of increased AMFR on transforming potential. The cells stably expressing high levels of AMFR as a result of transfection displayed a complete morphological change and acquired the ability to grow even in low serum. Furthermore, they were anchorage-independent for growth in soft agar and more motile in phagokinetic track assay. Interestingly, the enhanced expression of AMFR produced tumors in nude mice. Our findings provide a direct evidence that overexpression of the AMFR is associated with the acquisition of a transformation phenotype.