DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O4-Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4-O4 bond on processing by human DNA polymerase η (hPol η) was studied for oligonucleotides containing O4-methylthymidine, O4-ethylthymidine, and analogs restricting the O4-methylene group in an anti-orientation. Primer extension assays revealed that the O4-alkyl orientation influences hPol η bypass. Crystal structures of hPol η•DNA•dNTP ternary complexes with O4-methyl- or O4-ethylthymidine in the template strand showed the nucleobase of the former lodged near the ceiling of the active site, with the syn-O4-methyl group engaged in extensive hydrophobic interactions. This unique arrangement for O4-methylthymidine with hPol η, inaccessible for the other analogs due to steric/conformational restriction, is consistent with differences observed for nucleotide incorporation and supports the concept that lesion conformation influences extension across DNA damage. Together, these results provide mechanistic insights on the mutagenicity of O4MedT and O4EtdT when acted upon by hPol η.

4592 Background: Until ASCO 2007, the there was no proven systemic therapy for HCC, but dox was used often because of a response rate (RR) of up to 10%. We tested the effects of B, dox and B + dox in HCC cell lines and mouse models and found dox + B to be at least additive compared to dox alone. Methods: Eligible pts had biopsy proven HCC with no prior chemotherapy or hormonal therapy, ECOG PS 0–2, with measurable and biopsiable disease. Pts had adequate bone marrow function (lower levels allowed for splenomegaly), bilirubin ≤ 2.0 mg/dl, Child’s Pugh class A or B cirrhosis, INR ≤ 1.5, no peripheral neuropathy of grade 2 or higher and normal ejection fraction. Primary endpoint is RR. Null hypothesis = 10% RR. 40 pts were planned (37 eligible) to give 91% power to detect RR of 27% (7 responses) with 1-sided significance of 0.07. Secondary endpoints were toxicity, survival (OS) and progression-free survival (PFS). Dox (20 mg/m2 initially, then changed to 15mg/m2 due to heme toxicity) was given days 1, 8 (day 1 dox was omitted in cycle 1) and B (1.3mg/m2) (from NCI/CTEP) was given days 1, 4, 8 and 11 of a 21-day cycle. Serum was analyzed prior to treatment, cycle 1 day 8 and cycle 2 day 8 for changes in chemokines/cytokines (CKS). Results: 42 pts accrued, 39 confirmed eligible: 28 male, 11 female; 27 white, 9 African American, 3 other; PS = 0 :6, PS = 1: 28, PS =2: 5. 1 response (2.3%, 90% CI [0.1%, 11.6%]) was seen. 10 (25.6%) pts had stable disease, 17 pts (43.6%) had progression. The null response rate of 10% was not rejected (one-sided p-value = 0.98). Median OS was 5.7 months (90% CI [4.4, 7.9]) and PFS was 2.4 months (90% CI [2.1, 3.0]). 28/39 had grade (gr) 3 toxicity, 11 had gr 4. Most common toxicities were gr 3 leukocytes (11 pts), gr 3 platelets (8 pts) and gr 4 platelets (8 pts). Gr 3 fatigue occurred in 7 pts and gr 3 diarrhea in 5 pts. CKS evaluations showed changes in serum levels of GROα, MIP-1α, IL-6 and IL-8 in several pts. Conclusions: Dox + B did not show significant activity in HCC. The regimen was generally well-tolerated with mostly hematologic toxicity and no toxic deaths. B caused changes in several CKS levels in pts with HCC. We will analyze the lab data to correlate with clinical efficacy and toxicity prior to the ASCO 2008 meeting. No significant financial relationships to disclose.

The posttranscriptional modification of messenger RNA precursors by base deamination can alter profoundly the function of the encoded proteins. Adenosine deaminases that act on RNA (ADARs) are capable of catalyzing the site-specific conversion of adenosine to inosine in pre-mRNA transcripts, thereby affecting coding potential of mature mRNAs. ADAR1 is a member of ADAR1 family and is abundantly expressed in the brain and ubiquitously present in most tissues of the mammals. Alternatively spliced forms of ADAR1 cDNA were cloned from the mouse liver and the localization of these splicing forms were examined under a fluorescent microscope by fusing them to EGFP. The cDNAs of these splicing forms were cloned into a baculovirus expression vector and the recombinant protein of these splicing forms were produced in insect cells and purified with Ni-NTA resin. The double-stranded RNA editing activity of the recombinant proteins were determined and compared. The results showed four major splicing forms of ADAR1 were naturally present in mouse liver and consisted of cDNAs of 3.5 kb, 3.6 kb, 1.8 kb and 2.0 kb in length, respectively. These three splicing forms were La, Lb, Sa and Sb forms and found to have two distinct translation initiator codons. Furthermore, the four splicing forms of ADAR1 protein were localized to either the cytoplasm or nucleus of NIH 3T3 cells as shown by fluorescent microscopy. The double-stranded RNA editing activity of the four splicing forms also differ significantly, with the Lb form possessing the highest editing activity. In summary, four major mouse ADAR1 splicing forms are present naturally in the mouse liver with either cytoplasmic or nuclear localizations and widely varied editing activities, which indicate they might have different substrates and functions.

Adenosine deaminase acting on RNA (ADAR) in Drosophila and mammals has recently become the target of numerous investigations. It is now clear that this protein has a number of functions in the nervous system. Indeed, the mutation of ADAR in Drosophila ( dADAR) results in many pathological and physiological changes, such as sensitivity to hypoxia and neuronal degeneration. To understand the full scope of dADAR function, it is crucial to identify new dADAR targets. A polyclonal antibody against inosine was developed and used to enrich inosine-containing mRNAs. The efficiency of immunoaffinity purification was confirmed for the Q/R editing site of GluR-B pre-mRNA that has been edited by ADAR2 to generate inosines at the editing site. This approach was applied to enrich inosine-containing mRNAs from total mRNAs of wild-type and dADAR mutant flies, respectively. The enriched mRNA portion was then amplified and hybridized with Drosophila cDNA arrays. With this method, over 500 mRNAs were identified as potential dADAR targets by showing a higher amount in the enriched mRNA portion from wild-type flies than from dADAR mutant flies. The occurrence of A-to-G conversion in these mRNAs was further analyzed by comparing over 7,000 Drosophila cDNAs sequences with their genomic sequences. A final list of 62 candidates was generated from the overlap of the two approaches. Twelve genes from the final list were further examined by sequencing the RT-PCR products of these genes from wild-type and dADAR mutant flies. Seven of the 12 genes were proven to have A-to-G changes in the wild-type but not in mutant flies. We conclude that the combination of immunoaffinity enrichment of inosine-containing mRNA, DNA microarrays, and sequence comparison could facilitate the discovery of new dADAR substrates, which in turn allows us to better understand the targets of dADAR and the biological function of A-to-I RNA editing in flies.

ADAR1 is an RNA-specific adenosine deaminase that edits RNA sequences. We have demonstrated previously that different ADAR1 isoforms are induced during acute inflammation. Here we show that the mouse ADAR1 isoforms are differentially localized in cellular compartments and that their localization is controlled by several independent signals. Nuclear import of the full-length ADAR1 is predominantly regulated by a nuclear localization signal at the C terminus (NLS-c), which consists of a bipartite basic amino acid motif plus the last 39 residues of ADAR1. Deletion of the NLS-c causes the truncated ADAR1 protein to be retained in the cytoplasm. The addition of this sequence to pyruvate kinase causes the cytoplasmic protein to be localized within the nucleus. The localization of nuclear ADAR1 is determined by a dynamic balance between the nucleolar binding activity of the nucleolar localization signal (NoLS) in the middle of the protein and the exporting activity of the nuclear exporter signal (NES) near the N terminus. The NoLS consists of a typical monopartite cluster of basic residues followed by the third double-stranded RNA-binding domain. These signals act independently; however, NES function can be completely silenced by the NLS-c when a regulatory motif within the catalytic domain and the NoLS are deleted. Thus, the intracellular distribution of the various ADAR1 isoforms is determined by NLS-c, NES, NoLS, and a regulatory motif.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional process that amplifies the repertoire of protein production. Recently, the induction of this process through up-regulation of the editing enzyme RNA-specific adenosine deaminase 1 (ADAR1) was documented during acute inflammation. Here we report that the inflammation-induced up-regulation of ADAR1 involves differential production and intracellular localization of several isoforms with distinct RNA-binding domains and localization signals. These include the full-length ADAR1 (p150) and two functionally active short isoforms (p80 and p110). ADAR1 p80 starts at a methionine 519 (M519) due to alternative splicing in exon 2, which deletes the putative nuclear localization signal, the Z-DNA binding domain, and the entire RNA binding domain I. ADAR1 p110 is the mouse homologue of the human ADAR1 110-kDa variant (M246), which retains the second half of the Z-DNA binding domain, all RNA binding domains, and the deaminase domain. Additional variations are found in the third RNA binding domain of ADAR1; they are differentially regulated during inflammation, generating isoforms with different levels of activities. Studies in several cell types transfected with ADAR1-EGFP chimeras demonstrated that the p150 and p80 variants are localized in the cytoplasm and nucleolus, respectively. In agreement with this observation, endogenous ADAR1 was identified in the cytoplasm and nucleolus of mouse splenocytes and HeLa cells. Since the ADAR1 variants are differentially regulated during acute inflammation, it suggests that the localization of these variants and of A-to-I RNA editing in the cytoplasm, nucleus, and nucleolus is intracellularly reorganized in response to inflammatory stimulation.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of pre-mRNA catalysed by an RNA-specific adenosine deaminase (ADAR). A-to-I RNA editing has been previously reported in the pre-mRNAs of brain glutamate and serotonin receptors and in lung tissue during inflammation. Here we report that systemic inflammation markedly induces inosine-containing mRNA to approximately 5% of adenosine in total mRNA. Induction was the result of up-regulation of A-to-I RNA editing as both dsRNA editing activity and ADAR1 expression were increased in the spleen, thymus and peripheral lymphocytes from endotoxin-treated mice. Up-regulation of ADAR1 was confirmed in vitro in T lymphocytes and macrophages stimulated with a variety of inflammatory mediators including tumour necrosis factor-alpha and interferon-gamma. A late induction of RNA editing was detected in concanavalin A-activated splenocytes stimulated with interleukin-2 in vitro. Taken together, these data suggest that a large number of inosine-containing mRNAs are produced during acute inflammation via up-regulation of ADAR1-mediated RNA editing. These events may affect the inflammatory and immune response through modulation of protein production.

Aim To explore the changes of ADAR1 activity during mouse primary lymphocytic proliferation. Methods ① Synthetic dsRNA substrate labeled with 32 p-ATP was prepared by in vitro transcription using pBluescriptSK (+/-) vector containing a gene of α-tropomyosin; ② Lymphocytes were isolated from mouse spleen and lymph nodes, incubated in RPMI1640 with or without IL-2/ConA. The cells were harvested at different time points, and whole-cell extract was prepared so that the ADAR1 activities could be detected; ③The production of inosine in the synthetic dsRNA incubated with whole-cell extract was measured by TLC and radioautogram; ④Lymphocytic proliferation rate was detected by MTT colorimetry. Results ADAR1 activity was increased from 30 hours of incubation and reached higher level around 72 hours, which was consistent with the cell's proliferation curve. Conclusion Increased ADAR1 acitivity is first found during lymphocytic proliferation. ADAR1 might play an important role in the lymphocytic function.

目的 探索ADAR1活性升高对淋巴细胞细胞增殖的影响。方法 ①构建反转录病毒载体 :PCR从小鼠cDNA中放大ADAR1全长基因 ,连入反转录病毒MIEV载体 ,感染包装细胞后分泌病毒颗粒 ;②病毒颗粒感染T淋巴细胞CTLL2和B淋巴细胞A2 0 ,观察细胞增殖的变化 ;③MTT法测定细胞增殖率。结果 ADAR1/MIEV病毒感染淋巴细胞后 ,使细胞增殖速度减慢。结论 ADAR1在淋巴细胞发挥功能方面起有重要的作用 ,但哪些物质需要编辑以及具体机制如何 ,需要进一步研究。

We have recently identified the alpha-chemokine mob-1 as a highly inducible gene in several rat models of microvascular lung injury, whose expression was suppressed by inhibition of tumor necrosis TNF-alpha (TNF-alpha). This work provides further insight into the relationship between mob-1 and TNF-alpha in the development of lung injury assessed by pulmonary edema and leukosequestration. First, pulmonary mob-1 and TNF-alpha were upregulated in animals subjected to lung injury produced by the intratracheal administration of recombinant TNF-alpha and recombinant mob-1, respectively. Second, mob-1 inhibition by intratracheal anti-mob-1 antibody attenuated lung injury induced by recombinant TNF-alpha. Third, pretreatment with anti-TNF-alpha monoclonal antibody administered intratracheally abrogated recombinant mob-1-induced microvascular lung injury. In vitro, mob-1 and TNF-alpha increased each other's production in RAW 264.7 cells and mob-1 or TNF-alpha inhibition prevented endotoxin-induced upregulation of TNF-alpha or mob-1, respectively, from these cells. Together, these data suggest that mob-1 and TNF-alpha interact to promote lung inflammation.

Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury. To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)-induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon. ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours). Expression was localized to polymorphonuclear and monocytic cells. These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-gamma, a known inducer of ADAR1 in other cell systems. ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 microg/mL), live Escherichia coli (5x10(7) colony-forming units), or interferon-gamma (1000 U/mL). Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.