Abstract
This thesis investigates the pattern of gene expression interfered by chromium(VI) in CL3 human adenocarcinoma cells. A novel cDNA microarray assay was adopted to examine simultaneously the expression of 480 genes including those functioning in redox regulation, DNA repair, cell cycle regulation, signal transduction, apoptosis, and metastasis. Bacterial colonies containing putative genes were picked from the IMAGE library plates and amplified with polymerase chain reaction. The amplified cDNA fragments were spotted onto a positively charged nylon membrane using an arraying machine fitted with steel pins. CL3 cells were exposed to chromium(VI) in the presence or absence of ascorbate The first-strand cDNAs were l led with biotin using reverse transcriptase and the mRNAs extracted from the CL3 cells. The cDNA probes were used to hybridize to the genes on the nylon membrane. The gene expression patterns were determined by colorimetry and image analysis. Among the 480 genes, 11 were significantly induced by chromium(VI), including ATF-3, gadd45, gadd45b, gadd153, p21, 53BP1, neural cell adhesion molecule L1, ferredoxin reductase, heme oxygenase 1 (HO1), inhibitor of differentiation 2 (ID2), and heparin binding-EGF-like growth factor. Conversely, the expressions of 14 genes were significantly suppressed by chromium(VI), including vitronectin receptor a subunit, KAI1, DCC, NF-2, VLA-2, hMad-3, Smad1, Smad5, ATF-1, ATF-2, PTEN, pol A, PLAT, and Fadd. The results have indicated that most genes up-regulated by chromium(VI) participate in cell cycle arrest, apoptosis, and stress response regulation. In addition, most genes down-regulated by chromium(VI) are involved in tumor suppression and anti-metastasis. Whereas, chromium(VI) did not interfere with the expression of DNA repair genes. Ascorbate pre-treatment protected cell against chromium(VI)-induced cytotoxicity, and suppressed the HO1, ID2, ATF-3, gadd45b, gadd153, and p21 gene expression induced by chromium(VI). From these results, we may propose that the generation of cellular radical by chromium(VI) may be suppressed by ascorbate, thereby reduce cytotoxicity and the expression of genes involving in cell cycle arrest, apoptosis, and stress response regulation.