Bacterial mutagenicity and mammalian cell chromosomal and DNA damage in vitro assays were performed on a diesel exhaust particulate material (DPM) standard in two preparations: as an organic solvent extract, and as an aqueous dispersion in a simulated pulmonary surfactant. U.S. National Institute for Standards and Technology DPM SRM 2975 expressed mutagenic activity in the Salmonella reversion assay, and for in vitro genotoxicity to mammalian cells as micronucleus induction and as DNA damage in both preparations: as an acetone extract of the DPM mixed into dimethylsulfoxide, and as a mixture of whole DPM in a dispersion of dipalmitoyl phosphatidyl choline. Dispersion in surfactant was used to model the conditioning of DPM depositing on the deep respiratory airways of the lung. DPM solid residue after acetone extraction was inactive when assayed as a surfactant dispersion in the micronucleus induction assay, as was surfactant dispersion of a respirable particulate carbon black. In general, a given mass of the DPM in surfactant dispersion expressed greater activity than the solvent extract of an equal mass of DPM.

Because of their small size, the specific surface areas of nanoparticulate materials (NP), described as particles having at least one dimension smaller than 100 nm, can be large compared with micrometer-sized respirable particles. This high specific surface area or nanostructural surface properties may affect NP toxicity in comparison with micrometer-sized respirable particles of the same overall composition. Respirable particles depositing on the deep lung surfaces of the respiratory bronchioles or alveoli will contact pulmonary surfactants in the surface hypophase. Diesel exhaust ultrafine particles and respirable silicate micrometer-sized insoluble particles can adsorb components of that surfactant onto the particle surfaces, conditioning the particles surfaces and affecting their in vitro expression of cytotoxicity or genotoxicity. Those effects can be particle surface composition-specific. Effects of particle surface conditioning by a primary component of phospholipid pulmonary surfactant, diacyl phosphatidyl choline, are reviewed for in vitro expression of genotoxicity by diesel exhaust particles and of cytotoxicity by respirable quartz and aluminosilicate kaolin clay particles. Those effects suggest methods and cautions for assaying and interpreting NP properties and biological activities.

Diesel exhaust particulate material (DPM) was assayed for induction of chromosomal aberrations (CA), micronucleus (MN) formation, and 6-thioguanine-resistant (TG(r)) gene mutation in V79 cells as a dispersion in dipalmitoyl phosphatidylcholine (DPPC) in physiological saline, a simulated pulmonary surfactant. Filter-collected automobile DPM provided for the study was not organic solvent extracted, but was directly mixed into DPPC in saline dispersion as a model of pulmonary surfactant conditioning of a soot particle depositing in a lung alveolus. A statistically significant difference was found between treated and control groups at all concentrations tested in a CA assay. Assay for MN induction also gave a positive response: Above 50 mug/ml, the frequencies of micronucleated cells (MNC) were about 2 times higher than those in the control group. The forward gene mutation assay did not show a positive response when cells were treated with up to 136 mug DPM/ml for 24 h, as dispersion in DPPC in saline. Some comparison assays were run on direct dispersions of the DPM into dimethyl sulfoxide, with results equivalent to those seen with a DPPC-saline preparation: DPM in dimethyl sulfoxide (DMSO) was positive for MN induction but was negative for forward gene mutation in V79 cells. The positive clastogenicity results are consistent with other studies of DPM dispersed into DPPC-saline surfactant that have shown activity in mammalian cells for sister chromatid exchange, unscheduled DNA synthesis, and MN induction. The forward gene mutation negative results are consistent with studies of that assay applied to V79 cells challenged with DPM solvent extract.

Acetone extracts of engine exhaust particulate matter (PM) and of vapor-phase semi-volatile organic compounds (SVOCs) collected from a set of 1998-2000 model year normal emitter diesel engine automobile or light trucks and from a set of 1982-1996 normal emitter gasoline engine automobiles or light trucks operated on the California Unified Driving Cycle at 22 [degree]C were assayed for in vitro genotoxic activities. Gasoline and diesel PM were comparably positive mutagens for Salmonella typhimurium strains YG1024 and YG1029 on a mass of PM extract basis with diesel higher on a mileage basis; gasoline SVOC was more active than diesel on an extracted-mass basis, with diesel SVOC more active on a mileage basis. For chromosomal damage indicated by micronucleus induction in Chinese hamster lung fibroblasts (V79 cells), diesel PM expressed about one-tenth that of gasoline PM on a mass of extract basis, but was comparably active on a mileage basis; diesel SVOC was inactive. For DNA damage in V79 cells indicated by the single cell gel electrophoresis (SCGE) assay, gasoline PM was positive while diesel PM was active at the higher doses; gasoline SVOC was active with toxicity preventing measurement at high doses, while diesel SVOC was inactive at all but the highest dose.

Several studies have demonstrated the overexpression of certain eukaryotic translation factors in human cancer cell lines and in malignant tissues. In this study, with human cancer cell lines derived from lungs, breast, prostate, and skin, we have examined the expression profile of 36 translation factors consisting of 27 initiation factors, 8 elongation factors, and 1 termination factor. Translation initiation factors 2C2 and 4E1 and translation elongation factors 1A2 and 1δ were found overexpressed (2‐ to 2000‐fold) in many of the cancer cell lines compared to their corresponding normal cell lines. Among the translation factors analyzed, translation elongation factor 1A2 exhibited the most significant alteration in expression: 10‐ to 2000‐fold overexpression was noticed in nine out of ten cancer cell lines analyzed. Whether the overexpression of translation elongation factor 1A2 can be used as a potential tumor marker was tested with the cancer profiling array (BD Biosciences, Palo Alto, CA) consisting of 241 paired cDNA samples generated from 13 different cancer/noncancer tissue types. Overexpression of translation elongation factor 1A2 was noticed in several tumor tissue samples, most notably in the human colon cancer samples which exhibited at least a twofold overexpression among 35% of the samples analyzed. Besides colon, tumor samples derived from lungs, kidney, rectum, and ovary also exhibited more than a twofold overexpression of translation elongation factor 1A2 in at least 20% of the samples analyzed. These results indicate that human carcinogenesis is often associated with alterations in the expression of various translation factors especially the overexpression of eukaryotic translation elongation factor 1A2. © 2004 Wiley‐Liss, Inc.

OBJECTIVE:To study aberrant DNA methylation potentially resulting in changes in the expression of cancer-related genes as a possible epigenetic mechanism for cadmium carcinogenesis.METHODS:Genomic DNA isolated from CdCl(2)-transformed BALB/c-3T3 cells was digested with Mse1 (methylation non-sensitive) alone or with Mse1 and BstU1 (methylation sensitive). The resulting DNA was analyzed for aberrant methylation using PCR-based technique-Methylation-Sensitive Restriction Fingerprinting (MSRF). Several DNA fragments differentially methylated in the transformed cells identified by MSRF were confirmed by Southern hybridization analysis using the aberrantly methylated DNA fragments as the probes.RESULTS:Aberrant DNA methylation was identified in the transformed cells. DNA sequencing and sequence similarity analysis identified one of the aberrantly methylated DNA fragments as the p16 tumor suppressor gene.CONCLUSION:DNA hypermethylation is known to result in gene silencing, it appears that hypermethylation of p16 gene may represent a possible epigenetic mechanism for Cd-induced cell transformation and carcinogenesis.

OBJECTIVETo establish and identify CHO and COS7 cell lines transfected with TIF3cDNA.METHODSCHO and COS7 cell lines transfected with TIF3cDNA were established with plasmid (pcDNA3.1/V5-His-TOPO) as the vector, using calcium phosphate inductive transfection techniques and G418 cell selection protocols. Expression of the transfected gene was analyzed and identified using western blotting analysis. The non-transfected group and vector-transfected group were designed to compare with the target gene-transfected group.RESULTS3 out of 7 CHO cell lines transfected with TIF3cDNA expressed stably high levels of TIF3 encoded protein. Similarly, 2 out of 4 COS7 cell lines had significant over-expression of TIF3 encoded protein. Conclusion Three CHO and two COS7 cell lines transfected with TIF3cDNA have been successfully established. The cell lines are of great value for the future exploration of Cd compounds related oncogenens and for the studies of the other biological functions of the compounds.

目的对硅转化细胞基因组DNA异常甲基化进行研究,探讨硅的表遗传致癌机制.方法从结晶型硅(Si)转化BALB/c-3T3细胞中提取基因组DNA,经Mse1(甲基化非敏感性酶)单独消化或Mse1和BstU1(甲基化敏感性酶)联合消化,消化产物用甲基化敏感性内切酶指纹法(MSRF)进行分析,差异显示出异常甲基化基因片段,进一步将异常甲基化DNA片段亚克隆和序列测定,再与基因文库中的基因进行类比分析.结果发现硅转化细胞存在6条异常甲基化DNA(其中1条为高甲基化,5条有低甲基化现象),序列测定显示这些异常甲基化基因片段似乎来源于一些RNA转录和蛋白质翻译等基因家族.结论 DNA异常甲基化会导致基因表达激活或抑制,因此硅转化细胞基因组某些功能基因DNA异常甲基化导致的异常表达,可能间接是硅诱导细胞转化及其致癌作用的一种表遗传机制.

The molecular mechanisms potentially responsible for cadmium carcinogenesis were investigated by differential gene expression analysis of Balb/c-3T3 cells morphologically transformed with cadmium chloride. Differential display analysis of gene expression revealed overexpression of mouse Translation Initiation Factor 3 (TIF3; GenBank Accession Number AF 271072) and Translation Elongation Factor-1δ (TEF-1δ; GenBank Accession Number AF 304351) in the transformed cells compared with the control cells. The full length cDNAs for TIF3 and TEF-1δ were cloned and sequenced. Transfection of mammalian cells with an expression vector containing either TIF3 or TEF-1δ cDNA resulted in overexpression of the encoded protein. Overexpression of the cDNA-encoded TIF3 and TEF-1δ proteins in NIH3T3 cells was oncogenic as evidenced by the appearance of transformed foci capable of anchorage-independent growth on soft agar and tumorigenesis in nude mouse. Blocking the translation of TIF3 and TEF-1δ proteins using the corresponding antisense mRNA resulted in a significant reversal of the oncogenic potential of cadmium transformed Balb/c-3T3 cells as evidenced from the suppression of anchorage-independent growth on soft agar and diminished tumorigenesis in nude mouse. These findings demonstrate that the up-regulation of expression of TIF3 and TEF-1δ is a novel molecular mechanism responsible, at least in part, for cadmium carcinogenesis.

Objective TEF 1δ,mouse translation elongation factor\|1δ,is a novel gene cloned from CdCl 2\|transformed BALB/c\|3T3 cells,its GenBank Accession Number is AF304351.To test the biological functions of the novel gene in the cell transformation and carcinogenesis of cadmium.Methods The transfection technique,western analysis and cell transformation assay were used.Results The results have shown that,compared with untransfected and vector control,a 31 kDa protein was detected in Chinese hamster ovary (CHO) cells and in monkey kidney COS1 cells transfected with an expression vector (pcDNA3\^1/V5\|His\|TOPO Vector)containing TEF 1δ cDNA.Furthermore,transfection of NIH3T3 cells with an expression vector containing the TEF 1δ cDNA resulted in overexpression of the encoded protein and this was associated with cell transformation.Conclusion These findings demonstrate for the first time that the cell transformation and carcinogenesis induced by cadmium are due, at least in part,to the overexpression of TEF 1δ.It is probably a novel cadmium\|responsive proto\|oncogene.

The molecular mechanisms potentially responsible for cell transformation and tumorigenesis induced by cadmium, a human carcinogen, were investigated by differential gene expression analysis of BALB/c-3T3 cells transformed with cadmium chloride (CdCl(2)). Differential display analysis of gene expression revealed consistent overexpression of mouse translation initiation factor 3 (TIF3; GenBank accession number AF271072) in the cells transformed with CdCl(2) when compared with nontransformed cells. The predicted protein encoded by TIF3 cDNA exhibited 99% similarity to human eukaryotic initiation factor 3 p36 protein. A M(r) 36,000 protein was detected in cells transfected with an expression vector containing TIF3 cDNA. Transfection of NIH3T3 cells with an expression vector containing TIF3 cDNA resulted in overexpression of the encoded protein, and this was associated with cell transformation, as evidenced by the appearance of transformed foci exhibiting anchorage-independent growth on soft agar and tumorigenic potential in nude mice. Expression of the antisense RNA against TIF3 mRNA resulted in significant reversal of oncogenic potential of the CdCl(2)-transformed BALB/c-3T3 cells. Taken together, these findings demonstrate for the first time that the cell transformation and tumorigenesis induced by CdCl(2) are due, at least in part, to the overexpression of TIF3, a novel cadmium-responsive proto-oncogene.

目的: 构建TEF-1δ (mouse translation elongation factor-1δ) 的稳定表达系统.方法: 采用磷酸钙介导转染技术和G418细胞筛选法,以pcDNA3.1/V5-His-TOPO为表达载体,构建了TEF-1δ转基因CHO和COS7细胞系,Western Blot分析与鉴定表达蛋白.结果: 在10株转染和经G418反复筛选的CHO细胞系中,有3株(编码为COH-pcDNA3.1-TEF-1δ#3,#6,#14)具有高效稳定表达的TEF-1δ编码蛋白质(Mr约31×103),其余CHO细胞株的TEF-1δ蛋白质表达相对较弱或无表达.在4株转染和经G418反复筛选的COS7细胞系中,4株细胞(编码为COS7-pcCDNA3.1-TEF-1δ #4,#8,#14和#17)均有高效稳定的TEF-1δ编码蛋白表达,相应的无转染组及载体对照组的CHO和COS7细胞均无TEF-1δ蛋白质表达.结论: 这两类TEF-1δ转基因哺乳动物细胞稳定表达系统已成功构建与鉴定,该表达系统的建立对于TEF-1δ这一新基因的生物学功能研究,尤其是镉的致癌作用与致癌机制的研究有重要应用价值.

Recently we have identified, cloned, and characterized the mouse Translation Initiation Factor 3 (TIF3, GenBank Accession Number AF 271072) as a novel cadmium-responsive proto-oncogene. Presently, additional studies regarding the oncogenic potential of TIF3 have been carried out. Transfection of NIH3T3 cells with the pcDNA3.1 expression vector containing the TIF3 cDNA in the sense (5'-->3') orientation resulted in overexpression of the encoded 36 kDa protein. Transfection-mediated overexpression of TIF3 protein resulted in transformation of the cells as evidenced from the appearance of transformed foci. Cotransfection of the cells with a mixture of plasmid DNA consisting of TIF3 cDNA in the sense and in the antisense orientation resulted in significant inhibition of translation of the TIF3 protein. Antisense (3'-->5') TIF3 mRNA-mediated inhibition of translation of TIF3 protein, furthermore, resulted in inhibition of TIF3-mediated transformation of NIH3T3 cells as evidenced from the decrease in the number of transformed foci. These results further confirm that overexpression of TIF3 is oncogenic and the antisense TIF3 mRNA expression reverses its oncogenic potential.

TIF3 (GenBank Accession Number AF 271072) is identified as a novel cadmium- responsive proto-oncogene. In order to determine whether the antisense TIF3 reverses the oncogenic potential of Cd-transformed BALB/c-3T3 cells or not, a stable expression system of CdCl2-transformed BALB/c-3T3 cells with the expression vector containing TIF3 cDNA in the antisense orientation using calcium phosphate and G418 selection protocols is firstly established. Then, the reversal of the oncogenic potential of these cells is tested by soft agar and nude mouse tumorigenicity assay. The results demonstrated that expression of the antisense TIF3 in the CdCl2-transformed BALB/c-3T3 cells results in reversal of the transformed phenotype of the cells. This is evidenced by a 25%-70% decrease in the number of anchorage-independent colonies growing on soft agar and the significant reduced tumorigenic potential of cells in nude mice compared with the corresponding controls. In addition to a significant delay in the onset of appearance of tumors, a significant reduction in size and a 50.8%-55.1% decrease in weight of the tumors are also observed in the mice injected with the TIF3 antisense expressing cells compared with the corresponding controls. The results indicate that antisense TIF3 mRNA expression reverses its oncogenic potential of Cd-transformed BALB/c-3T3 cells and may have therapeutic potential to cancer induced by cadmium.

The molecular mechanisms of carcinogenesis by cadmium were studied using BALB/c-3T3 cell transformation and nude mouse tumorigenesis models. BALB/c-3T3 cells transformed with cadmium chloride were subcutaneously injected into nude mice to develop tumors and the cell lines derived from these tumors were used in the present study. The proto-oncogenes c-fos and c-jun were overexpressed in 100% (10 out of 10) of the cell lines, while a statistically significant overexpression of c-myc was observed in 40% (4 out of 10) of the cell lines. Analysis of tumor cells stained with fluorescent dyes specific for reactive oxygen species revealed that these cells possessed markedly higher levels of superoxide anion and hydrogen peroxide compared with the nontransformed cells. Similarly, the intracellular calcium level was higher in the tumor cells compared with the nontransformed cells. Overexpression of the proto-oncogenes in these cells was blocked by treating the cells with superoxide dismutase, catalase, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra acetoxy methyl ester (BAPTA/AM), which are scavengers of superoxide anion, hydrogen peroxide, and calcium, respectively. This confirmed that the overexpression of the proto-oncogenes in the tumor cells required elevated intracellular levels of reactive oxygen species and calcium. In addition to the scavengers of reactive oxygen species and calcium, inhibitors specific for transcription (actinomycin D), protein kinase C (RO-31-8220), and MAP kinase (PD 98059) also blocked the cadmium-induced overexpression of the proto-oncogenes in the tumor cells. Exposure of the nontransformed BALB/c-3T3 cells to 20 microM cadmium chloride for 1 h caused elevated intracellular levels of superoxide anion, hydrogen peroxide, and calcium, with corresponding increases in the expression levels of c-fos, c-jun, and c-myc. As in the case of the tumor cells, treating the nontransformed cells with the various modulators prior to their exposure to cadmium chloride resulted in inhibition in the expression of the proto-oncogenes. Based on these data, we conclude that the cadmium-induced overexpression of cellular proto-oncogenes is mediated by the elevation of intracellular levels of superoxide anion, hydrogen peroxide, and calcium. Further, the cadmium-induced overexpression of the proto-oncogenes is dependent on transcriptional activation as well as on pathways involving protein kinase C and MAP kinase.

The molecular mechanisms potentially responsible for cadmium-induced cell transformation and tumorigenesis were investigated using Balb/c-3T3 cells transformed with cadmium chloride. Differential display analysis of gene expression revealed consistent and reproducible overexpression of a transcript in the transformed cells compared with the nontransformed cells. The full-length cDNA corresponding to the differentially expressed transcript was cloned and was identified as mouse translation elongation factor-1 delta subunit (TEF-1 delta; GenBank accession number ). Nucleotide sequence analysis of TEF-1 delta cDNA revealed an open reading frame encoding the predicted protein of 281 amino acids and exhibited significant conservation with the corresponding protein of human, Xenopus laevis, and Artemia. The presence of a leucine zipper motif, characteristic of translation elongation factor-1 delta, was also found in the mouse TEF-1 delta. A 31-kDa protein was detected in eukaryotic cells transfected with an expression vector containing the TEF-1 delta cDNA. Overexpression of the TEF-1 delta protein by transfection was oncogenic in NIH3T3 cells as evidenced by the appearance of transformed foci exhibiting anchorage-independent growth and the potential to grow as tumors in nude mice. Blocking the translation of TEF-1 delta with antisense TEF-1 delta mRNA resulted in a significant reversal of the oncogenic potential of cadmium-transformed Balb/c-3T3 cells as evidenced from suppression in anchorage-independent growth and tumorigenesis in nude mice. Our findings demonstrate, for the first time, that the cell transformation and tumorigenesis induced by cadmium are due, at least in part, to the overexpression of TEF-1 delta, a novel cadmium-responsive proto-oncogene.

Differential gene expression was studied to understand the potential molecular mechanism responsible for cell transformation and tumorigenesis induced by beryllium. Cell lines were derived from tumors developed in nude mice injected subcutaneously with BALB/c-3T3 cells morphologically transformed with beryllium sulfate. Using the Atlas mouse 1.2 cDNA expression microarray, the expression profiles of 1176 genes, belonging to several different functional categories, were studied in the tumor cells as well as in the nontransformed control cells. Expression of 18 genes belonging to two functional groups was found to be consistently and reproducibly different (at least twofold) in the tumor cells compared with the control cells. The functional groups and the differentially expressed genes are as follows: The cancer-related genes (nine genes) were the ets-related transcription factor activated by ras, colony-stimulating factor, A-myb, sky, cot1, c-fos, c-jun, c-myc, and R-ras proto-oncogenes. The DNA synthesis, repair, and recombination genes (nine genes) were the DNA replication licensing factor MCM4, the DNA replication licensing factor MCM5, the DNA mismatch repair gene PMS2, the DNA excision repair gene, the DNA mismatch repair gene MSH2, the ultraviolet excision repair gene Rad23 DNA ligase 1, Rad51, and Rad52. The differential gene expression profile was confirmed with reverse transcription-polymerase chain reaction using primers specific for the differentially expressed genes. In general, expression of the cancer-related genes was upregulated, while expression of genes involved in DNA synthesis, repair, and recombination was downregulated in the tumor cells compared with the control cells. Using c-fos and c-jun, two of the differentially expressed genes, as model genes, we have found that in the nontransformed BALB/c-3T3 cells, the beryllium-induced transcriptional activation of these genes was dependent on pathways of protein kinase C and mitogen-activated protein kinase and independent of reactive oxygen species. These results indicate that beryllium-induced cell transformation and tumorigenesis are accompanied by and are possibly a product of alterations in expression of genes related to cancer and to DNA synthesis, repair, and recombination.