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Insulin-like growth binding protein-related protein 1 (IGFBP-rP1) has decreased expression in various tumors, but the role of IGFBP-rP1 in lung cancer has not yet been elucidated. In this study, we evaluated the IGFBP-rP1 expression in lung cancer cell lines and we found a reduced expression of IGFBP-rP1 at both mRNA and protein levels. In a tissue microarray containing 138 primary tumors analyzed by immunohistochemistry, 58 cases (42%) exhibited no expression of IGFBP-rP1, additionally, an association between IGFBP-rP1 expression and tumor gradings was found in squamous cell lung cancer. Neither deletion or gene rearrangement nor loss of heterozigosity was responsible for the inactivation of IGFBP-rP1. However, 5-aza-2'-deoxycytidine treatment restored the expression of IGFBP-rP1 in 3 out of 4 lung cancer cell lines. Sequencing of sodium bisulfite?treated genomic DNA from these 3 lung cancer cell lines revealed a heterogeneous methylation pattern in the region of exon 1 and intron 1. Stable transfection of IGFBP-rP1 full-length cDNA into a lung cancer cell line H2170 led to an increased protein expression of IGFBP-rP1. IGFBP-rP1 positive transfectants remarkably reduced the ability of colony formation in soft agar, suppressed the tumor growth rate in nude mice and increased the number of apoptotic cells as well as the expression level of casepase-3 compared to controls. Moreover, treatment with differentiation modulating agent 5-bromodeoxyuridine (BrdU) led to an enhanced expression of IGFBP-rP1 in lung cancer cells. Out data suggest that IGFBP-rP1 is a tumor suppressor possibly via DNA methylation and induction of apoptosis in human lung cancer.

Cell-Cell communication via gap junctions plays a key role in carcinogenesis and in growth control. One of the gap junction proteins, Connexin 26 (Cx26) was considered as tumor suppressor in various cancers. In our study, the expression of Cx26 was analyzed in human lung cancer. The reduced mRNA expression was observed in 17 lung cancer cell lines examined by Northern blot analysis and RT-PCR. In 138 primary carcinomas comprising all subtypes analyzed by immunohistochemistry, 85 cases (62%) exhibited no expression of Cx26, whereas in other 53 cases the Cx26 staining was positive (38%). Additionally, an association between Cx26 protein expression and higher grading of tumors was found in whole tumor samples (p =0.028) but no statistically significant correlations could be observed with tumor stage, tumor size and node status. In squamous cell carcinoma, tumors with higher stage and grading were linked to higher expression of Cx26 (p = 0.015 and 0.017, respectively). To explore the mechanism responsible for the downregulation of Cx26, we treated 2 lung cancer cell lines H2170 and H226 with the demethylation agent 5-aza-2'-deoxycytidine and found the reexpression of Cx26 mRNA. Methylation status of these 2 cell lines was further analyzed by PCR amplification of bisulfite modified DNA and sequencing. A heterogeneous methylation pattern turned out. Our results suggest the inactivation of Cx26 in lung cancer may be explained by promoter methylation.

Comparative genomic hybridization (CGH) was used to screen 22 esthesioneuroblastomas (ENB) from 12 patients including 12 primary tumors and 10 metastasis/recurrent lesions for chromosomal imbalances being the most extensive study so far. The analysis revealed a characteristic pattern consisting of deletions on chromosomes 3p and overrepresentations on 17q in up to 100% of cases. Other important alterations being detectable in more than 80% of cases were deletions on 1p, 3p/q, 9p, 10p/q along with overrepresentation on 17p13, 20p and 22q. Particularly striking was the pattern for chromosomes 3, 10 and 17q and 20 being affected almost exclusively by deletions or overrepresentations, respectively. Pronounced overrepresentations suggestive for high copy amplifications were seen on 1p34, 1q23-q31, 7p21, 7q31, 9p23-p24, 17q11-q22, 17q24-q25, 19, 20p, 20q13 and 22q13. Comparing tumor pairs from the same patient revealed a high concordance indicating clonality and confirming the genetic homogeneity of the tumor entity. The analysis of metastatic/recurrent lesions indicated a higher percentage of pronounced alterations, e.g., high copy DNA gains at 1q34-qter, 7q11, 9p23-p24, 9q34, 13q33-q34, 16p13.3, 16p11, 16q23-q24 and 17p13. The analysis furthermore suggested specific alterations, e.g., deletions of chromosome 11 and gains of 1p to be associated with metastasis formation and/or worse prognosis. Our results indicate that ENB is a distinct entity and provides criteria for its genetic distinction from other small round cell tumor types.

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. The expression of 22 clones from the 2 libraries was investigated by Northern blot analysis, and 86.4% (19/22) of them showed differential expression between Y-BE cells and HBEC. Although the Y-BE cells are nontumorigenic in nude mice, Comparative genomic hybridization (CGH) detected some DNA imbalances in Y-BE cells that were similar to lung cancer cells. Our data demonstrate that the studied cell line Y-BE and SSH is a reliable approach for identifying new genes that are associated with immortalization and early tumor development that may help to understand the pathogenesis of lung cancer.