We and others previously identified NKX2-1, also known as TITF1 and TTF-1, as a lineage-survival oncogene in lung adenocarcinomas. Here we show that NKX2-1 induces the expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between prosurvival PI3K-AKT and pro-apoptotic p38 signaling, in part through ROR1 kinase-dependent c-Src activation, as well as kinase activity-independent sustainment of the EGFR-ERBB3 association, ERBB3 phosphorylation, and consequential PI3K activation. Notably, ROR1 knockdown effectively inhibited lung adenocarcinoma cell lines, irrespective of their EGFR status, including those with resistance to the EGFR tyrosine kinase inhibitor gefitinib. Our findings thus identify ROR1 as an "Achilles' heel" in lung adenocarcinoma, warranting future development of therapeutic strategies for this devastating cancer.

Amplification and overexpression of the miR-17-92 microRNAs (miRNA) cluster at 13q31.3 has recently reported, with pointers to functional involvement in the development of B-cell lymphomas and lung cancers. In the present study, we show that inhibition of miR-17-5p and miR-20a with antisense oligonucleotides (ONs) can induce apoptosis selectively in lung cancer cells overexpressing miR-17-92, suggesting the possibility of 'OncomiR addiction' to expression of these miRNAs in a subset of lung cancers. In marked contrast, antisense ONs against miR18a and miR-19a did not exhibit such inhibitory effects, whereas inhibition of miR-92-1 resulted in only modest reduction of cell growth, showing significant distinctions among miRNAs of the miR-17-92 cluster in terms of their roles in cancer cell growth. During the course of this study, we also found that enforced expression of a genomic region, termed C2, residing 3 0 to miR-17-92 in the intron 3 of C13orf25 led to marked growth inhibition in association with double stranded RNA-dependent protein kinase activation. Finally, this study also revealed that the vast majority of C13orf25 transcripts are detected as Drosha-processed cleavage products on Northern blot analysis and that a novel polyadenylation site is present 3 0 to the miR-17-92 cluster and 5 0 to the C2 region. Taken together, the present findings contribute towards better understanding of the oncogenic roles of miR-17-92, which might ultimately lead to the future translation into clinical applications.

We developed infrared laser-evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock-mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.

microRNAs (miRNA) are small, endogenously expressed non-coding RNAs that are sequentially processed by Drosha and Dicer from primary transcripts, by negatively regulating the expression of protein-coding genes through either translational repression or RNA degradation. Their expression patterns are developmentally regulated and/or tissue specific, while altered expressions of certain miRNAs are frequently observed in human cancers, though the underlying regulatory mechanism is largely unknown. Herein, we show that Dicer expression was inversely correlated with expression levels of mature let-7 in a panel of human cancer cell lines, showing association with cell growth and cell cycle phases. Overexpression of let-7 significantly reduced the expression of Dicer at both the protein and messenger RNA levels, whereas antisense-mediated reduction of let-7 expression conversely increased Dicer at both levels. A luciferase assay using a reporter carrying a putative target site in the 3' untranslated region of Dicer revealed that let-7 directly affects Dicer expression. Downregulation of Dicer resulted in a reduced expression of mature let-7. Furthermore, overexpression of let-7 decreased the levels of expression of other mature miRNAs, while knockdown of let-7 increased those levels. Taken together, these findings strongly suggest the possible existence of a novel regulatory loop, in which let-7 may play a role as a key miRNA for implementing the tightly regulated, equilibrated state of Dicer and various miRNAs.

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We isolated active mutants in Saccharomyces cerevisiae DNA polymerase ␣ that were associated with a defect in error discrimination. Among them, L868F DNA polymerase ␣ has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase ␣. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase ␣-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase ␣ catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3 T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase , and the F34L mutant of S. cerevisiae DNA polymerase has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase ␣ is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.In all organisms with a DNA genome, the genome is replicated by DNA polymerases prior to cell division and one copy is transmitted to each daughter cell. In eukaryotic cells, the DNA polymerase ␣ (pol ␣)-DNA primase complex initiates DNA replication; DNA primase synthesizes a short RNA chain, and pol ␣ extends the RNA primer by ϳ30 deoxyribonucleotides. pol ε and pol ␦ continue DNA synthesis in a processive manner and complete the elongation stage of DNA replication (60).To ensure the survival of each daughter cell, DNA replication must be efficient and accurate. The insertion of the correct nucleotide depends in part on Watson-Crick base pair formation, but this mechanism is not sufficient to obtain high-fidelity DNA replication (23, 30). The observed high fidelity of chromosomal replication is achieved through mechanisms that enhance correct nucleotide insertion and that correct errors after they occur. Eukaryotic pol ␦ and pol ε remove misincorporated nucleotides with an intrinsic 3Ј-5Ј exonuclease activity (37, 38). Some evidence supports the idea of a physical and functional interaction between mismatch repair (MMR) proteins and PCNA, which is an accessory protein of pol ␦ and pol ε (1, 6, 18, 59). Furthermore, polymerase-associated exonucleases may participate in the same pathway that involves MMR (38,39,56). Disruption of this proofreading-MMR pathway causes types of cancers in both humans and mice (13,14,17).Little is known about error prevention and correction mechanisms that enhance the fidelity of DNA pol ␣. pol ␣ lacks 3Ј-5Ј exonuclease activity (36, 46), but it discriminates between correct and incorrect nucleotides during nucleotide incorporation and extension. Mutant polymerase characterization has provided some insight into the mechanism by which nucleotide insertion fidelity is achieved (7,26,41). However, it would be desirable to have a relevant e...