Increased expression and no mutation of the Flap endonuclease (FEN1) gene in human lung cancer.

The underlying molecular mechanisms leading to microsatellite alteration and mutations in human lung cancer remain unknown. Since Flap endonuclease1 (Fen1), which functions in the base excision repair system, has been shown to be involved in tumor progression of mouse models with microsatellite instability in a haplo-insufficient manner, we performed expression and mutation analyses for FEN1 in human lung cancer cell lines. Reverse transcriptase PCR analysis revealed that all 49 lung cancer cell lines (20 small cell lung cancers (SCLCs) and 29 non-small cell lung cancers (NSCLCs)) expressed FEN1. In addition, microarray analysis showed that FEN1 expression was elevated significantly by 1.65-fold (P=0.001) in SCLC cell lines compared to normal lung controls (normal human lung cultures and immortalized normal human bronchial epithelial cell lines). FEN1 protein was abundantly expressed in all 23 lung cancer cell lines (10 SCLCs and 13 NSCLCs) and was expressed at lower levels in three of four normal lung epithelial culture controls. Direct sequencing of genomic DNAs revealed no FEN1 mutation in seven SCLCs and nine NSCLCs. As part of this analysis we discovered and sequenced a FEN1 pseudogene (GenBank accession #AY249897) located at 1p22.2. This pseudogene is amplified from cDNA preparations contaminated with genomic DNA and must be taken into account in any FEN1 mutation analysis studies. Our results suggest that alterations of FEN1 are not likely to contribute to development of lung cancer.

Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo.

A group of candidate tumor suppressor genes (designated CACNA2D2, PL6, 101F6, NPRL2, BLU, RASSF1, FUS1, HYAL2, and HYAL1) has been identified in a 120-kb critical tumor homozygous deletion region (found in lung and breast cancers) of human chromosome 3p21.3. We studied the effects of six of these 3p21.3 genes (101F6, NPRL2, BLU, FUS1, HYAL2, and HYAL1) on tumor cell proliferation and apoptosis in human lung cancer cells by recombinant adenovirus-mediated gene transfer in vitro and in vivo. We found that forced expression of wild-type FUS1, 101F6, and NPRL2 genes significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle processes in 3p21.3 120-kb region-deficient (homozygous) H1299 and A549 cells but not in the 3p21.3 120-kb region-heterozygous H358 and the normal human bronchial epithelial cells. Intratumoral injection of Ad-101F6, Ad-FUS1, Ad-NPRL2, and Ad-HYAL2 vectors or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 tumor xenografts and inhibited A549 experimental lung metastases in nu/nu mice. Together, our results, coupled with other studies demonstrating a tumor suppressor role for the RASSSF1A isoform, suggest that multiple contiguous genes in the 3p21.3 120-kb chromosomal region may exhibit tumor suppressor activity in vitro and in vivo.

Transgenic expression of protein phosphatase 2A regulatory subunit B56gamma disrupts distal lung differentiation.

The distal epithelium of the developing lung exhibits high-level expression of protein phosphatase 2A (PP2A), a vital signaling enzyme. Here we report the discovery that in the lung, the PP2A regulatory subunit B56gamma is expressed in a discrete developmental period, with the highest protein levels at embryonic day (e) 17, but no detectable protein in the newborn or adult. By in situ hybridization, B56gamma was highly expressed in the distal epithelium of newly forming airways and in mesenchymal cells. In contrast, expression of B56gamma was quite low in the bronchial epithelium and vascular smooth muscle. Transgenic expression of B56gamma using the lung-specific promoter for surfactant protein C (SP-C) resulted in neonatal death. Examination of lungs from SP-C-B56gamma transgenic e18 fetuses revealed proximal airways and normal blood vessels, but the tissue was densely populated with epithelial-type cells and was devoid of normal peripheral lung structure. A component of the Wnt signaling pathway, beta-catenin, was developmentally regulated in the normal lung and was absent in lung tissue from B-56gamma transgenic fetuses. We propose that B56gamma is expressed at a particular stage of lung development to modulate PP2A action on the Wnt/beta-catenin signaling pathway during lung airway morphogenesis.

Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit.

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that regulates a wide variety of cellular processes. The enzymatic activity and intracellular localization of PP2A are determined by three distinct families of cellular regulatory subunits (B, B'', and B''). The B' subunit, also known as B56, is the most diverse, consisting of five isoforms (alpha, beta, gamma, delta, and epsilon). The gene encoding B56gamma has been designated as PPP2R5C and encodes three differentially spliced variants: B56gamma1, -gamma2, and -gamma3. However, conflicting chromosomal loci have been reported in human genomic databases. The original cytogenetic mapping placed the gene on chromosome 3p21.3, whereas subsequent studies using radiation hybrid analysis localized PPP2R5C to chromosome 14q. In this study, by radiation hybrid mapping, FISH analysis, BAC clone sequencing, and RT-PCR analysis, we show that the functional gene PPP2R5C exists at 14q32.2 and gives rise to three splicing variants, B56gamma1, -gamma2, and -gamma3, whereas a nonfunctional B56gamma1 pseudogene, PPP2R5CP, is present at 3p21.3. We also report the genomic organization of both the functional gene and the pseudogene.