LINE-1 and Alu elements are non-LTR retrotransposons, constituting together over 30% of the human genome and they are frequently hypomethylated in human tumors. A relationship between global hypomethylation and genomic instability has been shown, however, there is little evidence to suggest active role for hypomethylation-mediated reactivation of retroelements in human cancer. In our study, we examined by Pyrosequencing the methylation levels of LINE-1 and Alu sequences in 48 primary nonsmall cell carcinomas and their paired adjacent tissues. We demonstrate a significant reduction of the methylation levels of both elements (p 5 7.7 3 10 214 and 9.6 3 10 27 , respectively). The methylation indices of the 2 elements correlated (p 5 0.006), suggesting a possible common mechanism for their methylation maintenance. Genomic instability was measured utilizing 11 fluorescent microsatellite markers located on lung cancer hot-spot regions such as 3p, 5q 9p, 13q and 17p. Hypomethylation of both transposable elements was associated with increased genomic instability (LINE, p 5 7.1 3 10 25 ; Alu, p 5 0.008). The reduction of the methylation index of LINE-1 and Alu following treatment of 3 lung cell lines with 5-aza-2 0 -deoxycitidine, consistently resulted in increased expression of both elements. Our study demonstrates the strong link between hypomethylation of transposable elements with genomic instability in non-small cell lung cancer and provides early evidence for a potential active role of these elements in lung neoplasia. As demethylating agents are now entering lung cancer trials, it is imperative to gain a greater insight into the potential reactivation of silent retrotransposons in order to advance for the clinical utilization of epigenetics in cancer therapy. ' 2008 Wiley-Liss, Inc.Key words: LINE-1; Alu; hypomethylation; genomic instability; lung cancer LINE-1 elements are autonomous non-LTR retrotransposons comprising almost 21% of the human genome. 1 Although most of these elements are inactive, mostly due to 5 0 truncations, there are a number of 80-100 intact LINE-1 elements that remain capable of retrotransposition. 2,3 The full length human LINE-1 retrotransposon is about 6 kb consisting of a 5 0 UTR, two open reading frames (ORF 1 and ORF2), and a 3 0 UTR with a functional polyadenylation signal and a variable length poly-A tail. 1 The bicistronic mRNA of LINE-1 is generated by an internal promoter at the 5 0 UTR region. ORF1 encodes for p40, a 40 kDa RNA-binding protein with cis preference for LINE-1 RNA, while ORF2 encodes for a 150 kDa protein with 3 conserved domains. 4 LINE-1 retrotransposition is undertaken with the help of both ORF1 and ORF2 proteins through a target-primed reverse transcription mechanism (TPRT). 2 In normal human cells, transcription and retrotransposition of LINE-1 is suppressed by a variety of control mechanisms including methylation, siRNAs and transcription regulators. [4][5][6] As a result, cells manage to protect themselves from a number of adverse effects exerted by LINE-1 ...

BACKGROUND:The UHRF1 gene possesses an essential role in DNA methylation maintenance, but its contribution to tumor suppressor gene hypermethylation in primary human cancers currently remains unclear. METHODS: mRNA expression levels of UHRF1, DNMT1, DNMT3A, DNMT3B, and E2F1 were evaluated in 105 primary nonsmall cell lung carcinomas by quantitative polymerase chain reaction. The methylation status of CDKN2A and RASSF1 promoters was examined by pyrosequencing. UHRF1 was knocked down by short hairpin RNA in A549 lung adenocarcinoma cells. RESULTS: All 4 genes were overexpressed in a coordinated manner in the lung tumor tissues, and their expression correlated with that of E2F1. Higher UHRF1 expression in tumor tissues correlated with the hypermethylation of CDKN2A (P ¼ .005) and RASSF1 promoters (P ¼ .034), and the relationship with a combined epigenotype was even stronger (P ¼ 2.3 Â 10 À4 ). When UHRF1 was knocked down in A549 lung adenocarcinoma cells, lower methylation levels of RASSF1, CYGB, and CDH13 promoters were observed. Also, UHRF1 knockdown clones demonstrated reduced proliferation and decreased cell migration properties. CONCLUSIONS: Our data demonstrate that UHRF1 is a key epigenetic switch, which controls cell cycle in nonsmall cell lung carcinoma through its ability to sustain the transcriptional silencing of tumor suppressor genes by maintaining their promoters in a hypermethylated status. Thus, UHRF1 should be considered, along with DNMTs, among the potential targets for cancer treatment and/or therapeutic stratification.

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