Risk of benign meningioma after childhood cancer in the DCOG-LATER cohort:contributions of radiation dose, exposed cranial volume, and age

Pediatric cranial radiotherapy (CrRT) markedly increases risk of meningiomas. We studied meningioma risk factors with emphasis on independent and joint effects of CrRT dose, exposed cranial volume, exposure age, and chemotherapy. The Dutch Cancer Oncology GroupLong-Term Effects after Childhood Cancer (DCOG-LATER) cohort includes 5-year childhood cancer survivors (CCSs) whose cancers were diagnosed in 19632001. Histologically confirmed benign meningiomas were identified from the population-based Dutch Pathology Registry (PALGA; 19902015). We calculated cumulative meningioma incidence and used multivariable Cox regression and linear excess relative risk (ERR) modeling. Among 5843 CCSs (median follow-up: 23.3 y, range: 5.052.2 y), 97 developed a benign meningioma, including 80 after full- and 14 after partial-volume CrRT. Compared with CrRT doses of 119 Gy, no CrRT was associated with a low meningioma risk (hazard ratio [HR] = 0.04, 95% CI: 0.010.15), while increased risks were observed for CrRT doses of 2039 Gy (HR = 1.66, 95% CI: 0.833.33) and 40+ Gy (HR = 2.81, 95% CI: 1.306.08). CCSs whose cancers were diagnosed before age 5 versus 1017 years showed significantly increased risks (HR = 2.38, 95% CI: 1.394.07). In this dose-adjusted model, volume was not significantly associated with increased risk (HR full vs partial = 1.66, 95% CI: 0.863.22). Overall, the ERR/Gy was 0.30 (95% CI: 0.03unknown). Dose effects did not vary significantly according to exposure age or CrRT volume. Cumulative incidence after any CrRT was 12.4% (95% CI: 9.8%15.2%) 40 years after primary cancer diagnosis. Among chemotherapy agents (including methotrexate and cisplatin), only carboplatin (HR = 3.55, 95% CI: 1.627.78) appeared associated with meningioma risk. However, we saw no carboplatin dose-response and all 9 exposed cases had high-dose CrRT. After CrRT 1 in 8 survivors developed late meningioma by age 40 years, associated with radiation dose and exposure age, relevant for future treatment protocols and awareness among survivors and physicians

Human Argonaute proteins: Analysis of endonucleolytic activity and endogenous phosphorylation sites

Argonaute (Ago) proteins interact with small non-coding RNAs, which guide them to complementary target RNAs to mediate gene silencing. The domain organization of Ago proteins clearly reflects their function. The ends of the small RNA are bound by the Mid and PAZ domains and the structure of the PIWI domain closely resembles that of the endonuclease RNase H. Indeed, some Ago proteins can cleave perfectly complementary target RNAs. Here, the endonucleolytically inactive human Ago proteins Ago1, Ago3, and Ago4 were mutated to yield minimally changed, catalytically active proteins. The generation of these mutants revealed several features that are important for Ago cleavage activity, in particular two regions in the N-terminal domain and two structural elements in the catalytic domain. The identification of these structural requirements contributes essentially to our understanding of Ago-mediated silencing activity. Ago proteins not only cleave their targets. In case of partial complementarity between small RNA and target RNA, they recruit additional factors to mediate gene silencing. Here, predominant mechanisms are translational repression and reduction of target stability. Considering the wide spectrum of gene regulation emanating from Ago proteins, it is important to understand how the proteins are regulated themselves. Here, post-translational modifications, in particular phosphorylations, of Ago proteins were analyzed by mass spectrometry. Multiple endogenous phosphorylation sites of Ago2 and its paralogues were detected. Among them are sites, which are organized in phosphorylation clusters. The data generated in this thesis represents a valuable resource for future analyses of Ago proteins and their modifying enzymes, which will provide insights into the interplay of small RNA-guided gene silencing and signaling pathways. The phosphorylation sites analyzed here were identified on endogenous Ago proteins. To provide large amounts of endogenous protein, a novel, peptide-based purification strategy was established. Ago Affinity Purification by Peptides (Ago-APP) is based on the interaction of Ago proteins with a conserved binding partner, a member of the GW182 protein family. Therefore, the approach purifies all four human Ago proteins and can even be applied species-independently. Ago-APP represents a powerful tool for the characterization of diverse Ago complexes at the levels of protein, small RNA, and target RNA

Argonaute Family Protein Expression in Normal Tissue and Cancer Entities.

The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease

The Slicer Activity of ARGONAUTE1 is Required Specifically for the Phasing, Not Production, of Trans-Acting Short Interfering RNAs in Arabidopsis

ARGONAUTE1 (AGO1) mediates posttranscriptional silencing by microRNAs (miRNAs) and short interfering RNAS (siRNAs). AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets, but current models also highlight the roles of slicing in formation of siRNAs and siRNA-AGO1 complexes. miRNA-guided slicing is required for biogenesis of phased, trans-acting siRNAs (tasiRNAs), whose cleaved precursor fragments are converted to double-stranded RNA by RNA-dependent RNA polymerase 6 (RDR6). In addition, unwinding of duplex siRNA bound to AGO1 requires passenger strand cleavage in vitro. In this study, we analyze how mutation of four metal ion-coordinating residues of Arabidopsis thaliana AGO1 affects slicer activity in vitro and siRNA function in vivo. We show that while all four residues are required for slicer activity, they do not contribute equally to catalysis. Moreover, passenger strand cleavage is required for assembly of active AGO1-siRNA complexes in vivo, and many AGO1-bound siRNAs are trimmed in the absence of slicer activity. Remarkably, seedlings defective in AGO1 slicer activity produce abundant siRNAs from tasiRNA loci in vivo. These siRNAs depend on RDR6 and SUPPRESSOR OF GENE SILENCING3, but unlike wild-type tasiRNAs, they are unphased. These results demonstrate that slicing is solely required for phase definition of tasiRNAs, and they strongly support recruitment of RDR6 by AGO1 rather than by cleavage fragments

Argonaute protein distribution in melanoma and non-melanoma cell lines.

<p>(A) AGO protein percentage distribution of each AGO to total AGO protein amount in non-melanoma cell lines CaCo2, HepG2, SW1353, MCF7, HeLa and melanoma cell lines Mel Ju, Mel Im, Mel Wei, Mel Ei and Mel Ho derived from AGO-APP. (B) Total protein amount determination of each AGO enriched by AGO-APP in CaCo2, HepG2, SW1353, MCF7, HeLa, Mel Ju, Mel Im, Mel Wei, Mel Ei and Mel Ho. (C) Average AGO protein amount of each AGO in non-melanoma compared to melanoma cell lines. The reduced AGO concentration in melanoma cell lines compared to non-melanoma cell lines is only significant for AGO2. ** = p<0.01 (D) AGO1 western blot analysis and corresponding (E) Western blot quantification of melanoma cell lines (Mel Ju, Mel Im, Mel Wei, Mel Ei, Mel Ho) and non-melanoma cell lines (CaCo2, SW1353, MCF7, HeLa, HepG2). The two additional values in the AGO1 western blot quantification illustrate the average AGO1 concentration in melanoma and non-melanoma cell lines. Quantification was done relative to Actin in the respective blot.</p

Argonaute gene expression in different human healthy tissues compared to NHEMs.

<p>Relative (A) AGO1, (B) AGO2, (C) AGO3 and (D) AGO4 mRNA expression in NHEMs (derived from different cultivation passages (P4-P6) from three different donors respectively), skin (derived from three different tissue samples) and heart, kidney, bone marrow, fetal brain, bowel, thymus, uterus, trachea, brain, muscle, bone marrow, liver, fetal liver, brain and fetal brain (derived from a total RNA bank and shown as technical replicates). (E) The compilation of percentage distribution of each AGO to the total AGO amount in the respective cell line or tissue.</p

Argonaute gene expression in different melanoma and non-melanoma cell lines.

<p>Relative (A) AGO1, (B) AGO2, (C) AGO3 and (D) AGO4 mRNA expression in melanoma cell lines derived from primary tumors (Mel Ei, Mel Juso, Mel Ho and Mel Wei) and metastases (Mel Ju, Mel Im, SkMel28 and Hmb2) and other non-melanoma cell lines (HeLa, CaCo2, PLC, Jurkat, Hep3b, SW1353 and MCF7). Each point shows the measurement of one independently derived cDNA sample. Bars show mean and S.D. (E) The compilation of percentage distribution of each AGO to the aggregate AGO amount. (F) Entire mRNA expression of all four AGOs compared to actin in melanoma cell lines derived from primary tumors or metastases and in other non-melanoma cell lines.</p

Oligonucleotide sequences and qRT-PCR conditions.

<p>Oligonucleotide sequences and qRT-PCR conditions.</p