Abstract
Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).
Highlights
Neoplastic transformation of human fibroblasts and epithelial cells is thought to result from the sequential acquisition of genetic and/or epigenetic alterations in specific genes [1]
Phenotypic characteristics of UE6E7T-3 cell line. This laboratory has previously demonstrated that aneuploidy, the loss of one copy of chromosome 13, arises in the immortalized human mesenchymal stem cells (hMSC) line UE6E7T-3 after prolonged culture [21,22]
This study shows that the hMSC line, UE6E7T-3, immortalized with the hTERT gene in combination with the HPV-16 E6/E7 genes, gradually transformed during prolonged culture
Summary
Neoplastic transformation of human fibroblasts and epithelial cells is thought to result from the sequential acquisition of genetic and/or epigenetic alterations in specific genes [1]. The results of these studies suggest that the transformation of human cells in vitro depends upon functional alterations in four to six genes. These alterations include changes in genes involved in telomere maintenance (to extend replicative lifespan), disruption of tumor suppressor pathways, and activation of oncogenes [2,3,4,5,6,7,8,9,10]. The transformation of normal human fibroblasts requires the co-expression of MYC, RAS, and hTERT together with the functional loss of the RB, PTEN, and p53 tumor suppressor pathways. A review by Duesberg and colleagues suggests that aneuploidy, in which a cell contains an abnormal number of chromosomes, is the primary cause of, and driving force behind, tumorigenesis: they state that aneuploidy results in an imbalance of gene expression, leading to the initiating event that initiates the transformation of normal cells [11]
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