No abstract

In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed "Inverted Repeat Binding Protein (IRBP) 18" and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.T ransposable elements contribute significantly to the organization and evolution of all eukaryotic genomes. Recent estimates of transposon content within the Drosophila melanogaster genome are between 5% and 10%, and in humans over half the genome is composed of mobile elements (1, 2). Although many of these elements, including the Drosophila P-element transposon, are still active (3), the cellular mechanisms used to combat the genotoxic effects of DNA double-strand breaks (DSBs) generated by transpositional recombination are not fully understood. The Drosophila P-transposable element provides an excellent model for understanding the ancient mechanisms used by the cell to counteract newly invading parasitic mobile DNA elements (4).The P-element transposon is a mobile DNA element that spread through wild populations of D. melangaster ∼100 y ago after most common laboratory strains were isolated (5, 6). P elements were identified by studying a genetic syndrome called "P-M hybrid dysgenesis." It was observed that males from wild populations (P strains) crossed to females from isolated laboratory stocks (M strains) yielded progeny that had germline mutations, temperature-sensitive sterility, and atypical male recombination (6). Reciprocal crosses yielded phenotypically normal progeny. The P element was shown to be the causative agent of these so-called P-M hybrid dysgenesis phenotypes by molecular analyses showing that P elements were present in variable locations in P strains yet totally absent from most M strains (7,8).The Drosophila P-element transposon encodes a GTP-dependent site-specific DNA transposase/integrase family enzyme (9, 10). At each end of the P-element transposon are perfect 31-bp terminal inverted repeats (TIRs), 11-bp internal inverted repeats that serve as enhancers of transposition, and internal 10-bp transposase binding sites (11-13) (Fig. 1A). The P-element transposase catalyzes DNA cleavage within the 31-bp TIRs to create 17-nt 3′ single-strand extensions at both the donor site and the transposon ends (14, 15)...

IGF-I has been implicated in the pathogenesis of human cancer. We sought to establish a role for IGF-I in the regulation of telomerase, an enzyme critically involved in cancer cell immortalization. Telomerase activity was assayed in LAPC-4, PC-3, and DU-145 prostate cancer cell lines treated with and without IGF-I/IGF-I analogs. Relative expression of human telomerase reverse transcriptase (hTERT) mRNA and protein was determined by quantitative RT-PCR and Western immunoblot, respectively. IGF-I stimulated baseline telomerase activity in all three cell lines, ranging from 2- to 10-fold (P < 0.05). Enhancement was noted at IGF concentrations as low as 10 ng/ml and was maximal at 100 ng/ml. Stimulation was noted by 0.5 h, was maximal by 8 h, and persisted to 48 h. A similar 3-fold enhancement (P < 0.01) was noted in response to Long-R3 IGF-I, but not in response to [Ala(31),Leu(60)]IGF-I. Pretreatment with the Akt kinase inhibitor wortmannin abolished the stimulatory IGF effect, whereas blockade of MAPK activity did not. Lastly, IGF-I provoked a 2-fold increase in hTERT mRNA and protein expression (P < 0.01). In summary, IGF-I clearly stimulates telomerase activity in prostate cancer cells through a dual mode of action, including early rapid effects probably involving phosphorylation of hTERT by Akt and later up-regulation of hTERT expression.

IGF-binding proteins (IGFBPs) have multiple cellular effects, which occur by both IGF-dependent and -independent mechanisms. IGFBP-2 is involved in the regulation of both normal and carcinogenic cell growth. To further understand the actions of IGFBP-2, we carried out a yeast two-hybrid screen to search for intracellular partner proteins using a human prostate cDNA library. We isolated Pim-1-associated protein-1 (PAP-1)-associated protein-1 (PAPA-1) as an IGFBP-2-binding protein, whose expression and subcellular localization is regulated by both IGFBP-2 and androgens. Coimmunoprecipitation and glutathione S-transferase pull-down assay confirmed the interaction in vitro, and confocal microscopy showed the colocalization of IGFBP-2 and PAPA-1 in the nucleus. Suppression of PAPA-1 by small interfering RNA treatment enhanced the growth-promoting effect of IGFBP-2. Conversely, IGFBP-2-promoted bromodeoxyuridine incorporation into LNCaP cells was abrogated by the simultaneous overexpression of myc-hPAPA-1. Mouse embryonic fibroblasts from IGFBP-2 knockout mouse showed diminished growth activity compared with wild type, and expression of FLAG-mPAPA-1 decreased cell proliferation in IGFBP-2 knockout, but not control mouse embryonic fibroblasts. These studies suggest that the growth-promoting role of IGFBP-2 in prostate cancer is inhibited by its intracellular interaction with PAPA-1.