Identification of differentially expressed genes in immortalized human bronchial epithelial cell line as a model for in vitro study of lung carcinogenesis

Abstract

Suppression subtractive hybridization (SSH) was applied to identify differentially expressed genes in the SV40LT immortalized human bronchial epithelial cell line Y-BE, with normal human bronchial epithelial cells (HBEC) as a control. Two cDNA libraries of up-and downregulated genes were generated, comprising 218 known genes and 131 unknown genes in total. Non-small cell lung cancer (NSCLC) is believed to originate from bronchial epithelial cells and accounts for about 75% of lung cancers, 1 which is the leading cause of cancer death all over the world. It is of great importance to elucidate the mechanisms involved in human bronchogenic carcinogenesis at the cellular and molecular levels. It has been demonstrated that lung tumorigenesis undergoes a process of multiple and sequential morphological and molecular changes. 2 The mechanisms of pathogenesis of lung cancer are poorly understood, however, mostly due to the difficult in vivo analysis of precancerous lesions and the lack of suitable experimental models.We showed previously that immortalization of a human bronchial epithelial cell line (Y-BE) by simian virus 40 (SV40) large T antigen (LT) transfection did result in altered cell proliferation and apoptosis, as well as other transforming phenotypes, such as reduced dependence on growth factor, increased resistance to seruminduced differentiation and anchorage independence for growth in soft agar. Some of the phenotype alterations, like low dependence on EGF, resistance to serum-induced differentiation and decrease in cisplatinum-induced apoptosis, became obvious only in later passages of the cells. 3,4 Although various cellular and molecular aberrations could be observed, the cells even at late passages are non-tumorigenic in nude mice. Notably, cells from another human bronchial epithelial cell line (M-BE), which was also immortalized by SV40-LT in our lab and showed similar cytogenetic and molecular genetic alterations during immortalization, can form typical precancerous epithelial lesions (e.g., squamous metaplasia, mild and severe dysplasia) in a recently developed in vivo animal model for lung carcinogenesis, 4 implicating that these immortalized cell lines represent preneoplastic stages of carcinogenesis of bronchial epithelium and not a malignant lung carcinoma.Using the methods of comparative genomic hybridization (CGH) and suppression subtractive hybridization (SSH), we now present a variety of cytogenetic and molecular genetic alterations of Y-BE cells detected during the immortalization. Our data demonstrated that the cell line Y-BE could be used as a relevant model for bronchogenic carcinogenesis, and that further study on those new genes unveiled by SSH may cast light on the understanding of pathogenesis of lung cancer. MATERIAL AND METHODS Cell linesThe immortalized human bronchial epithelial cell line Y-BE, derived from outgrowths of biopsy tissue from a non-cancerous female individual, was established by SV40 large T antigen transfection in our lab. 5 The cells were cultivated in serum-free ...

Expression of targeting protein for xklp2 associated with both malignant transformation of respiratory epithelium and progression of squamous cell lung cancer.

“…The gradually increased clusters of TPX2 positively stained cell nuclei were observed along with the progression from the squamous metaplasia, to dysplasia, and carcinoma in situ. The expressive status of TPX2 in the precancerous lesions was consistent with that detected in the immortalized bronchial epithelial cell lines (e.g., M-BE and Y-BE) that were considered as in the ''precancerous state'' (24,25). Previous studies indicated that TPX2-overexpressing cells exhibited DNA polyploidy, implying that excess TPX2 may inhibit nuclear division (26) and that aneuploidy could be detected at the transition from the squamous metaplasia to the dysplasia (27) and frequency of aneuploidy increased with the progressive atypia in the respiratory epithelium of lung cancer patients (28).…”

Identification of genes expressed differentially in an in vitro human lung carcinogenesis model

“…Some of the genes identified in the present study were consistent with a previous study with SV40 large T antigen-transformed bronchial epithelial cells (KRT14/19, S100A2/A8/A11, ANAX8, GJB2, ITGA6, GSTP1, UBE2C and PTMS). 28 The changes in the expression of KRT5/14/19, S100A11, ANAX8, and LAMC2 in our study were similar to the findings with normal breast luminal epithelial cells transformed with SV40 large T antigen. 29 These similarities raised the possibility that some of the changes in gene expression may be related to the infection with Adeno/SV40 large T antigen and not necessarily with the immortalization process.…”

Identification of genes differentially expressed in human primary lung squamous cell carcinoma

“…Among the LSCC library, genes such as AKR1B10, AKR1C1, AKR1C3, KRT6C, BPAG1, TPX2, GSTM3, TFRC, NME1, GPNMB and PPP1CB have been described to be overexpressed in lung tumours [12][13][14][15][16][17][18][19][20]. Of note, GNAS, FN1, GLTP, RAC1, and APLP2 were also identified as upregulated genes related to SV40T immortalized human bronchial epithelial cells [6] and/or primarily cultured human lung SCC tumour cells [7]. Gene ontology analysis was subsequently performed on the differentially expressed genes in the LSCC library.…”

“…In our study, overexpression of TBL1XR1 was also validated at the protein level in the lung tumour tissues and cancer cell lines. In particular, the overexpression of TBL1XR1 was revealed in the immortalized bronchial epithelial cell line (M-BE) that is considered ''precancerous'' [6]. This suggested that deregulation of TBL1XR1 might be an early event during lung cancer development.…”

“…SSH has been successfully applied to a wide variety of malignant diseases [4,5]. In our previous studies using SSH, genes that were differentially expressed in SV40T immortalized human bronchial epithelial cells or primarily cultured lung SCC tumour cells in comparison with their normal counterparts were described [6,7]. However, either the SV40T immortalized cells or primarily cultured cells differ from primary tissue cells that have been removed from their in vivo environment and selected for growth characteristics at in vitro culture conditions, thus bringing into question the clinical relevance of these findings.…”