As one high risk etiological factor of OSCC, HPV type 16 was previously used to immortalize normal oral epithelial cells, while this immortalized cell line could not form tumors in nude mice.15 However, the cells acquired tumorigenesity through subsequent exposure to B (a) P followed by continued passaging in vitro.16 So, the biological events occurring during the malignant transformation process are crucial in oncogenic research.
In the present study, hybridization of mRNA-derived probes to cDNA microarray allowed us to generate expression profiles for thousands of genes simultaneously and to identify the molecular mechanisms underlying a continuous transformation process during which an immortalized cell line acquired increasing tumorigenesity through B (a) P exposure. We distinguished genes that played roles in different stages of the transformation process successfully through a Venn diagram. Most parts of the differentially expressed genes focused on the stage of HB-56p versus HIOEC, during which cells possess tumorigenesity initially, though the tumor contained mostly benign contents. This finding indicates that genes associated with this process may play a crucial role in the transformation process.
The differentially expressed genes were then annotated using Gene Ontology. Most of these genes involved in macromolecule metabolism, signal transduction and regulation of cellular physiological process, which may be the common physiological process in transformed cells. These changed genes mainly possessed molecular functions of transition metal ion binding, adenyl nucleotide binding, kinase activity, and transcription factor or cofactor activity, suggesting that genes with these functions might be crucial in the transformation process. Their protein products were mainly integral to membranes, localized in the nucleus and cytoskeleton, which depicted the crucial sites of change in transformed cells and the cytoskeleton associated genes partly explained the changes in cell shape of HB cells compared with HIOEC cells.
B (a) P first required metabolic activation by phase I enzymes and then were subjected to detoxification by phase II enzymes. Phase I enzymes were encoded by the cytochrome P450 (CYP450s) gene family, whereas phase II enzymes included the glutathione-S-transferases (GSTs).19 The metabolism formed a proposed ultimate carcinogen, this diol epoxide metabolite of B (a) P was capable of forming stable DNA-adducts leading to mutations and oxidative damage in target organs.20 In addition, genes involved in the metabolism of B (a) P did not change significantly in this study as it did in other documents.11,12,19,20 In this study, CYP24A1, a member of CYPs, was up-regulated in set B with a fold change of 3.071 and GSTT2, a member of GSTs, was up-regulated in set D with a fold change of 2.367, whereas genes of GSTA4, MGST2, CYP1B1 and CYP27B1, were down-regulated in set B. One reason may be that the metabolism of B (a) P was only an initial factor of carcinogenesis, with the deprivation of B (a) P and the continuous passaging in vitro, these metabolism associated genes would lose their influence on the downstream event.
We did not find genes which were up or down-regulated in neoplastic cells compared with normal cells and immortalized cells in the study of Rey,14 such as cyclophilin A, tumor necrosis factor receptor associated protein p67, c-myc promoter binding protein (MBP1), antileukoproteinase SKALP and heat shock protein 90 alpha, which were differentially expressed in this study.
Five genes were consistently down-regulated during the transformation process of both HB-56p versus HIOEC and HB-96p versus HB-56p. Gap junction protein alpha 1 (GJA1, also named connexin 43), plays a role in cell communication. Overexpression of GJA1 through retroviral delivery in breast tumor cells did result in a dramatic suppression of tumor growth when the cells were implanted into the mammary fat pad of nude mice. 22 Loss of GJA1 in human tumors might play an important role in the dysregulation of normal growth control. 23 Plasma membrane localization and formation of channels were not required for growth inhibition by GJA1, whereas nuclear localization of C-terminal portion of GJA1 may exert effects on gene expression and growth.24 Snail homolog 2 (SNAI2, also named SLUG) was a negative regulator of transcription from RNA polymerase II promoter and a ces-1-related zinc finger transcription factor gene with antiapoptotic activity.25 Over expression of SNAI2 may promote tumorigenesis through increased resistance to programmed cell death in cell lines from human breast carcinoma and melanoma.26 One possible explanation for this discrepancy may be related to the different origin of the epithelial cells. Interferon induced transmembrane protein 1 (IFITM1) attended in negative regulation of cell proliferation, which associates with other proteins at the cell surface, formed complex relaying growth inhibitory and aggregation signals.27
In summary, we have identified genes differentially expressed in oral epithelial cells transformed by B (a) P. These gene subsets can potentially help in better understanding the development mechanisms associated with OSCC derived from B (a) P exposure and IGFBP3 may play a potential role in the initiation of oral cancer related with B (a) P exposure.
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