Preliminary Applications Of Mouse Toxicology Gene Chip On The Investigations Of Tumor-promoting Chemicals

It is general realized that the progress of cancer caused by chemicals is a long time, multistage, multi-gene and multi-pathogeny. The multistage carcinogenesis is involved with three steps including initiation, promotion and progression, and chemical carcinogens influence tumor development in many ways by acting as initiators, promoters or progressors, while the tumor promoters play a major role in the multistage carcinogenesis. It has long been elucidated that the tumor promoters can induce tumor development in previously initiated cells by causing their selective expansion, and this effect is reversible at the early time of tumor promotion, thus early studies on the identification and inhibition of the effects of tumor promoters have presaged modern chemoprevention in humans. On the other hand, Toxicologists employ a battery of tests to identify chemicals with potential carcinogenicity and these methods have been proved to be costly and labor intensive. It is apparent that alternative testing approaches must be developed to improve the identification of potential carcinogens including tumor-promoting agents. Additionally, the mechanisms of actions of tumor promotes are poorly understood, in a general sense, it is promised that the mechanisms of tumor promotion are also involved with multiple genes and signalling pathways, more and more genes have been found to play major role in this process, thus it is not effective to address the complex mechanism of tumor-promoting action with traditional expression-based technologies, such as northern blots, in situ hybridization or RNase protection assays, which examine gene expression changes for only a few genes at a time.cDNA chip technology, which can be used to analyze changes in genome-wide patterns of gene expression, is a new methodological advance that may revolutionize the way of investigations for some toxicological problems, such as carcinogenesis. This technology enable the simultaneous monitoring of thousands of genes expression changes in one experiment, it will be useful to identify the characteristic gene expression patterns, what so called gene fingerprint, for specific toxicant or carcinogen. The potential uses of gene chip in toxicology involve to identify chemicals of the basis of tissue specific patterns of gene expression by establishing molecular signature for chemical exposure, and to elucidate themechanisms of action of environmental agents through the identification of gene expression network. Till now, some investigations have already proved that the gene chip technology could be successfully applied to detect and identify the specific gene expression of potential carcinogens, that also indicates this technology will be useful in the investigation of tumor promotion. In order to find out clues for further identifying the genes related to cell transformation and the characteristic gene expression patterns reflected specific tumor promoter(TPA, OA or CdCl2), nine clones induced by MNNG and different tumor promoter in the two-stage BALB/c 3T3 cell transformation test were used to detect the gene expression profiles with mouse toxicology gene chips. Besides, for understanding the molecular mechanisms of the cell transformation promoted with tumor promoters(TPA, OA or CdCia), cells treated with different tumor promoter in the two-stage BALB/c 3T3 cell transformation test were harvested in each time point during the promotion stage, and gene expression profiles were detected with mouse toxicology gene chips. This experimental study was composed of four parts: (1) Establishing the in vitro two-stage BALB/c 3T3 cells transformation test with MNNG as initiator, TPA, OA or CdC^ as promoter respectively; (2) Establishing and evaluating the technique of fabrication and detection for mouse toxicology gene chip; (3) Analysis of gene expression profiles for transformed cell clones induced by different tumor promoter; (4) Analysis of gene expression profiles for BALB/c 3T3 cells treated with different tumor promoter during the promotion stage. The main results were summed up as follows:1. The two-stage transformation test of BALB/c 3T3 cells were established with MNNG as initiator, TPA, OA or CdCl2 as promoter respectively. The promoters significantly enhanced the transformation of cells initiated with ll^g/ml MNNG, whereas high dose promotes failed to induce transformation without pretreatment of MNNG At the same time, nine clones transformed with MNNG and different promoter were choosed and cultured for the further investigation, and all these clones had the character of anchorage-independent growth in soft agar tests.2. 1796 genes related to eight biological functions were choosed for the construction of the mouse toxicology gene chip, and the corresponding database was established at the same time. Preliminary investigations indicated this toxicology chip designed by ourselves could applied in the identification and evaluation for toxicant actions including tumor promoting effect.3.The gene fragments used to fabricate the gene chip were copied from NIA mouse cDNA clone sets, and the result of gene fragments proliferation showed that 95.12% of the PCR products had strong and single band. Moreover, five batches of slides had good images in the selective examination with Picogreen dye method. These results indicated that the proliferation of target genes and the fabrication of chips had been fulfilled successfully.4. Preliminary toxicological tests proved the validity of the chip hybridization and detection technology established in our lab according to the reference, and the effectivity of the locally mean normalization method to analyze the original data.5. Several methods were applied to verify the reliability and reproducibility of the gene chip data. The results showed that there was no nonspecific hybridization in the negative control spots; Good reproducibility could achieved among the repetitive genes in a chip; Different batches of chips had instant quality and reproducibility; Dye swap labeling had advantages in reducing the dye errors; Finally the real-time RT-PCR method validated most of the data come from chip hybridization. These results proved the accuracy of the gene chip data.6. Gene expression profiles of transformed clones induced by different promoter were screened with mouse toxicology gene chips. 236 genes were found to differentially expressed in nine transformed clones compared with normal cells, and the classification of gene functions indicated that the largest three functional gene groups of each transformed clone were involved with cell growth/maintenance, signal transduction, transcriptional regulation or metablism.7. 236 differentially expressed genes in the transformed clones were hierarchically clustered and nine transformed clones were separated into three main branches in concordance with the promoter used to induce them. The result suggested that transformed clone maybe has specific tumor promoter-associated gene expression profile.8. Two clusters of genes showed similar expression patterns among all of the transformed clones, one cluster consisted genes up-regulated(23), and the other cluster consisted genes down-regulated(26). The expression change of these genes suggested a possibility that they may play a role in the cell transformation.9. Several clusters of genes showed characteristic expression patterns that highlighted the differences among the different promoter induced transformed clones. One cluster consisted 6 genes up-regulated in TPA-induced clones, one cluster consisted 14 genes up-regulated inOA-induced clones, and the other cluster consisted 18 genes up-regulated in CdCl2-induced clones. These genes differentially expressed among different promoter induced clones were expected to be related with the molecular mechanisms specific to a promoter.10. Gene expression profiles of cells treated with different promoter during the promotion stage of BLAB/c 3T3 cells transformation test were detected with mouse toxicology gene chip. The numbers of differentially expressed genes influenced by each promoter were 120 in TPA, 177 in OA and 139 in CdCl2, and the classification of gene functions showed that the largest functional gene group induced by each promoter was similar and involved with cell growth/maintenance. Combined with previous result of cell growth curve, these results suggested that the promoting effects of TPA, OA and CdC^ in the early time of promotion are related to cell growth arrest or apoptosis induction.11. Further analysis for the functions of differentially expressed genes involved with cell proliferation or apoptosis suggested the possibilities that TPA could influence the transcriptional expression of some genes mainly related to the ras and P53 signal transduction pathways and ultimatedly result in the cell growth arrest, and the possibility of apoptosis induction also exists. In addition, OA could influence the expression of genes mainly related to the mitochondrial apoptosis pathway and then induce cell apoptosis. while CdCh could influence the expression of genes related to mitochondrial apoptosis pathway and Fas-ligand apoptosis pathway simultaneously and then induce cell apoptosis.12. A group of genes related to the antioxidation were differentially expressed after the treatment of TPA, OA or CdCl2. This result suggested the possibility that during the promotion stage, promoter could cause the unbalance between oxidation and antioxidation in cells, and the created reactive oxygen species may act as secondary messengers to initiate the apoptosis.13. There were same differentially expressed genes induced by TPA, OA and CdCl2, among these genes, SPP1 was up-regulated mainly in the middle and late stage of promotion. Functional analysis indicates that SPP1 mediates tumor metastasis and invasion, funcions in the regulation of cell cycle progression and prevention of apoptosis.