Diesel exhaust particulate matter dispersed in a phospholipid surfactant induces chromosomal aberrations and micronuclei but not 6-thioguanine-resistant gene mutation in V79 cells.

Diesel exhaust particulate material (DPM) was assayed for induction of chromosomal aberrations (CA), micronucleus (MN) formation, and 6-thioguanine-resistant (TG9 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 microg/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 microg 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.

In vitro genotoxicity of exhaust emissions of diesel and gasoline engine vehicles operated on a unified driving cycle.

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.

Expression profile of eukaryotic translation factors in human cancer tissues and cell lines.

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 1delta 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.

Up-regulation of expression of translation factors--a novel molecular mechanism for cadmium carcinogenesis.

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-1delta (TEF-1delta; GenBank Accession Number AF 304351) in the transformed cells compared with the control cells. The full length cDNAs for TIF3 and TEF-1delta were cloned and sequenced. Transfection of mammalian cells with an expression vector containing either TIF3 or TEF-1delta cDNA resulted in overexpression of the encoded protein. Overexpression of the cDNA-encoded TIF3 and TEF-1delta 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-1delta 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-1delta is a novel molecular mechanism responsible, at least in part, for cadmium carcinogenesis.

[Effect of aberrant DNA methylation on the expression of cancer-related genes in Cd-transformed cells].

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.

Antisense inhibition of translation initiation factor 3 reverses its oncogenic potential.

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.

Molecular cloning and functional analysis of 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.

[Antisense TIF3 reverses the oncogenic potential of CdCl2-transformed BALB/c-3T3 cells].

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.

Oncogenic potential of mouse translation elongation factor-1 delta, a novel cadmium-responsive proto-oncogene.

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.