Vector-only Control Research Articles

Live vaccine consisting of attenuated Salmonella secreting and delivering Brucella ribosomal protein L7/L12 induces humoral and cellular immune responses and protects mice against virulent Brucella abortus 544 challenge

The present study employs the Brucella abortus L7/L12 antigen in a Salmonella secretion platform and investigates its ability to induce protective immune responses against wild type challenge in BALB/c mice. The highly conserved L7/L12 open reading frame was PCR amplified from B. abortus and cloned into a prokaryotic expression vector, pJHL65, directly under the beta-lactamase secretory signal. The plasmid constructs pJHL65::L7/L12 was then transformed into an attenuated Salmonella Typhimurium strain, JOL1800 (∆lon, ∆cpxR, ∆asd, and ∆rfaL), and protein secretion was verified by Western blot. Three mice groups were inoculated with either phosphate-buffered saline (PBS), vector-only control, or the vaccine strain secreting L7/L12 antigen. Assessment of humoral and cell-mediated immune responses revealed successful elicitation of Brucella antigen-specific Th1 and Th2 immune responses that were significantly higher than PBS and vector control groups. The immune responses were confirmed by splenocyte proliferation assay, flow cytometry analysis for CD4+ and CD8+ markers, and RT-PCR based cytokine profiling upon restimulation with L7/L12 purified antigen. Results indicate that immunization with Salmonella secreting L7/L12 antigen demonstrated significant enhancement of cell-mediated immune (CMI) responses in immunized mice. The overall effectiveness of the immunization was evaluated by challenging with virulent B. abortus that revealed significant reduction in Brucella colonization in spleen and liver tissues in Salmonella L7/L12 immunized mice. Delivery of Brucella protective antigen L7/L12 using the Salmonella secretion system can effectively accomplish immunogenic advantages of both Salmonella and L7/L12 to derive robust CMI responses and induce humoral immunity to protect against Brucella infection in the mouse model.

Oncogenesis of CRLF2 Overexpression and Effect of JAK2 Inhibitor in CRLF2 Overexpressed B-Cell Acute Lymphoblastic Leukemia

Backgroud: Cytokine receptor-like factor 2 (CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate the proliferation of early B cells. However, the diect oncogenic effect of CRLF2 overexpression in acute lymhpoblastic leukemia (ALL) is far yet to be clarified. Here, we explored the effect of CRLF2 overexpression on cell proliferation and the effect of the novel JAK2 inhibitor on B-ALL cells with CRLF2 overexpression. Methods: The 83 patients with newly-diagnosed ALL (56 B-cell and 27 T-cell ALL; range from 14 to 77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 21 normal bone marrow subjects were enrolled as controls. The qPCR method is developed for detection CRLF2 expression and the CRLF2 overexpression was determined with a cutoff value more than the highest sample of normal bone marrow control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. CRLF2 F232C gain-of-function mutant which we previously reported or CRLF2 were expressed in Nalm6 and 697 B-ALL cells with lentiviral transduction. WST-1 cell proliferation assay and in vitro clonogenic assay were performed upon JAK2 inhibitor (BBT594) treatment. Nalm6-CRLF2-luc, Nalm6-F232C-luc, and Nalm6-vector-luc cells were injected via tail vein into the NSG mice. The leukemia engraftment was monitored once a week by living imaging. Results: The expression of CRLF2 in patietns with ALL was significantly higher than the normal control (P<0.0001). Patients with CRLF2 overexpression had a significantly higher WBC count (53*10^9/L vs. 29.5*10^9/L, P=0.041). Survival analysis showed that the patients with CRLF2 overexpression had a worse EFS and OS, the differences were statistically significant (11 months vs. 26 months, P=0.043 and 15 months vs. 32 months, P=0.015). Also, the CRLF2 expression is determined with flow cytometry after staining with FITC-CRLF2 antibody in 28 samples. The correlation analysis was performed on the CRLF2 expression detected by qPCR and flow cytometry, respectively. A significant positive correlation of the two methods was observed(r=0.957, P<0.0001). These data not only indicate that CRLF2 overexpression is a marker of poor outcome, but also reveal the qPCR might be a simple and quick method for screening CRLF2 overexpression in the clinic compared to flow cytometry which is commonly used. We further found that expression of CRLF2 or CRLF2 F232C mutant into Nalm6 and 697 B-ALL cells dramatically increase the CRLF2 mRNA level, which is 69 times than vector-only control. Moreover, CRLF2 or CRLF2 F232C significantly promotes the cell proliferation of Nalm6 and 697 cells compared to vector only (P<0.001). In addition, JAK2 inhibitor (BBT594) treatment showed the significant dose-dependent cell proliferation arrest and clonogenic inhibition in CRLF2 or CRLF2 F232C overexpressed Nalm6 and 697 cells compared to vector-only control. Furthermore, in vivo we observed the 5-fold higher signal intensity of leukemia engraftment in the mice injected with Nalm6-CRLF2-luc or Nalm6-F232C-luc compared to that of Nalm6-vector-luc control 1-3 weeks after the injection(P<0.001). The Nalm6-CRLF2-luc and Nalm6-F232C-luc infiltrations were observed in bone marrow, central nervous system, liver and spleen of the mice. Conclusion: We showed that CRLF2 overexpression could enhance the proliferation and infiltration of human B-ALL cells, and for the first time indicated that JAK2 inhibitor could suppress the cell proliferation and clonogenesis of the CRLF2 overexpressed B-ALL cells. Our data provide direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with JAK2 inhibitor. Disclosures No relevant conflicts of interest to declare.

The cancer-associated, gain-of-function TP53 variant P152Lp53 activates multiple signaling pathways implicated in tumorigenesis

TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L) in TP53 at the very end of its DNA-binding domain (DBD) in a sample from an Indian oral cancer patient. Although the P152Lp53 DBD alone bound to DNA, the full-length protein completely lacked binding ability at its cognate DNA motifs. Interestingly, P152Lp53 could efficiently tetramerize, and the mutation had only a limited impact on the structure and stability of full-length p53. Significantly, when we expressed this variant in a TP53-null cell line, it induced cell motility, proliferation, and invasion compared with a vector-only control. Also, enhanced tumorigenic potential was observed when P152Lp53-expressing cells were xenografted into nude mice. Investigating the effects of P152Lp53 expression on cellular pathways, we found that it is associated with up-regulation of several pathways, including cell-cell and cell-extracellular matrix signaling, epidermal growth factor receptor signaling, and Rho-GTPase signaling, commonly active in tumorigenesis and metastasis. Taken together, our findings provide a detailed account of the biochemical and cellular alterations associated with the cancer-associated P152Lp53 variant and establish it as a gain-of-function TP53 variant.

Novel Genes Encoding Hexadecanoic Acid Δ6-Desaturase Activity in a Rhodococcus sp.

cis-6-Hexadecenoic acid, a major component of human sebaceous lipids, is involved in the defense mechanism against Staphylococcus aureus infection in healthy skin and closely related to atopic dermatitis. Previously, Koike et al. (Biosci Biotechnol Biochem 64:1064-1066, 2000) reported that a mutant strain of Rhodococcus sp. produced cis-6-hexadecenoate derivatives from palmitate alkyl esters. From the mutant Rhodococcus strain, we identified and sequenced two open reading frames present in an amplified 5.7-kb region; these open reading frames encoded tandemly repeated Δ6-desaturase-like genes, Rdes1 and Rdes2. A phylogenetic tree indicated that Rdes1 and Rdes2 were different from previously known Δ6-desaturase genes, and that they formed a new cluster. Rdes1 and Rdes2 were each introduced into vectors and then expressed separately in Escherichia coli, and the fatty acid composition of the transformed cells was analyzed by gas chromatography and mass spectrometry. The amount of cis-6-hexadecenoic acid was significantly higher in Rdes1- or Rdes2-transformed E. coli cells (twofold and threefold, respectively) than in vector-only control cells. These results showed that cis-6-hexadecenoic acid was produced in E. coli cells by the rhodococcal Δ6-desaturase-like proteins.

Mycobacterium tuberculosis PE13 (Rv1195) manipulates the host cell fate via p38-ERK-NF-κB axis and apoptosis.

PE/PPE family proteins are mycobacteria unique molecules, named after their N-terminal conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains. Mycobacterium tuberculosis (Mtb) PE family gene encoded cell surface proteins are previously reported to be involved in virulence and interaction with host. To explore the role of a novel PE member (PE13, Rv1195), M. smegmatis was used as surrogate host. The study showed that Rv1195 was a cell wall associated protein. Rv1195 can enhance the survival of recombinants under stress conditions such as H2O2, SDS, low pH. This is largely due to the upregulated transcription of Rv1195, since diverse stresses can increase the promoter activity of Rv1195 gene, consistent with enhanced survival within macrophages. Ms_Rv1195 infection also increased the production of interlukin-6 (IL-6) and IL-1β from macrophages, while decreased the secretion of suppressor of cytokine signaling 3 (SOCS3) in comparison with the vector-only control. The cell death was also precipitated by the Ms_Rv1195 infection. Inhibitors treatment showed that the p38-ERK-NF-κB axis was involved in the Rv1195 triggered change of IL-6 and IL-1β expression. In summary, we showed that PE13 (Rv1195) is a new PE family member actively engaged in the interaction between Mycobacterium and host, signaling through p38-ERK-NF-κB axis and apoptosis.

The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii.

Overexpression of the resistance-nodulation-cell division-type efflux pump AdeABC is often associated with multidrug resistance in Acinetobacter baumannii and has been linked to mutations in the genes encoding the AdeRS two-component system. In a previous study, we reported that the Asp20→Asn amino acid substitution in the response regulator AdeR is associated with adeB overexpression and reduced susceptibility to the antimicrobials levofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. To further characterize the effect of the Asp20→Asn substitution on antimicrobial susceptibility, the expression of the efflux genes adeB, adeJ, and adeG, and substrate accumulation, four plasmid constructs [containing adeR(Asp20)S, adeR(Asn20)S, adeR(Asp20)SABC, and adeR(Asn20)SABC] were introduced into the adeRSABC-deficient A. baumannii isolate NIPH 60. Neither adeRS construct induced changes in antimicrobial susceptibility or substrate accumulation from that for the vector-only control. The adeR(Asp20)SABC transformant showed reduced susceptibility to 6 antimicrobials and accumulated 12% less ethidium than the control, whereas the Asn20 variant showed reduced susceptibility to 6 of 8 antimicrobial classes tested, and its ethidium accumulation was only 72% of that observed for the vector-only construct. adeB expression was 7-fold higher in the adeR(Asn20)SABC transformant than in its Asp20 variant. No changes in adeG or adeJ expression or in acriflavine or rhodamine 6G accumulation were detected. The antimicrobial susceptibility data suggest that AdeRS does not regulate any resistance determinants other than AdeABC. Furthermore, the characterization of the Asp20→Asn20 substitution proves that the reduced antimicrobial susceptibility previously associated with this substitution was indeed caused by enhanced efflux activity of AdeB.

Bcl2 inhibition of mitochondrial DNA repair.

BackgroundAccumulation of mitochondrial DNA (mtDNA) damage could enhance the frequency of mitochondrial mutations and promote a variety of mitochondria-related diseases, including cancer. However, the mechanism(s) involved are not fully understood.MethodsQuantitative extended length PCR was used to compare mtDNA and nDNA damage in human lung H1299 cells expressing WT Bcl2 or vector-only control. mtAPE1 endonuclease activity was analyzed by AP oligonucleotide assay. mtDNA mutation was measured by single molecule PCR. Subcellular localization of Bcl2 and APE1 was analyzed by subcellular fractionation.ResultsBcl2, an anti-apoptotic molecule and oncoprotein, effectively inhibits the endonuclease activity of mitochondrial APE1 (mtAPE1), leading to significant retardation of mtDNA repair and enhanced frequency of mtDNA mutations following exposure of cells to hydrogen peroxide (H2O2) or nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, a carcinogen in cigarette smoke). Inversely, depletion of endogenous Bcl2 by RNA interference increases mtAPE1 endonuclease activity leading to accelerated mtDNA repair and decreased mtDNA mutation. Higher levels of mtAPE1 were observed in human lung cancer cells than in normal human bronchial epithelial cells (i.e. BEAS-2B). Bcl2 partially co-localizes with APE1 in the mitochondria of human lung cancer cells. Bcl2 directly interacts with mtAPE1 via its BH domains. Removal of any of the BH domains from Bcl2 abolishes Bcl2’s capacity to interact with mtAPE1 as well as its inhibitory effects on mtAPE1 activity and mtDNA repair.ConclusionsBased our findings, we propose that Bcl2 suppression of mtDNA repair occurs through direct interaction with mtAPE1 and inhibition of its endonuclease activity in mitochondria, which may contribute to enhanced mtDNA mutations and carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1594-1) contains supplementary material, which is available to authorized users.

MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS Te-Fu Tsai, Ji-Fan Lin, Yi-Chia Lin, Kuang-Yu Chou, Hung-En Chen, and Thomas I.S. Hwang Te-Fu TsaiTe-Fu Tsai More articles by this author , Ji-Fan LinJi-Fan Lin More articles by this author , Yi-Chia LinYi-Chia Lin More articles by this author , Kuang-Yu ChouKuang-Yu Chou More articles by this author , Hung-En ChenHung-En Chen More articles by this author , and Thomas I.S. HwangThomas I.S. Hwang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.738AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES mTOR is recognized as an important target in many cancer types. The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be insensitive to RAD001 and was shown to regulate the prosurvival kinase AKT by phosphrylation. We previously showed that the expression level of miR-99a was down-regulated in human bladder tumor tissues compared to their adjacent normal tissues. Since mTOR is predicted to be a potential target of miR-99a, we investigate the anti-cancer activity of miR-99a in bladder cancer cells. METHODS Vectors that express miR-99a were transfected into human bladder, prostate, embryonic kidney and cervical cancer cells. Expression level of miR-99a was monitored by miRNA Q-PCR. 3'-UTR of mTOR was constructed downstream of a luciferase gene, and luciferase reporter assays were performed to verify the direct binding of miR-99a to mTOR transcripts. The mRNA and protein expression level of mTOR were measured by Q-PCR and Western blot, respectively. Cell viability of miR-99a transfected cells was detected by WST-1 and colony formation assays. Inhibition of mTOR complex 1 and 2 (mTORC1 and mTORC2) signaling was monitored by detecting the phosphrylation of S6K and Akt using Western blot. Induction of autophagy was accessed by the expression of LC3-II marker protein. RESULTS Transfection of miR-99a expressing vector elevated the expression level of miR-99a up to 4.5-fold in cells compared to vector-only control. The function of matured miR-99a was confirmed by luciferase reporter assays. The level of mTOR RNA and protein were decreased in miR-99a transfected cells. Dual inhibition of mTORC1 and mTORC2 was confirmed by immunoprecipitation (IP) of mTOR associated Rictor and Raptor, and the decreased phosphrylation of S6K and Akt in miR-99a transfected cells. The LC3-II protein was accumulated in miR-99a transfected cells compared to monk transfected control, suggesting that inhibition of mTOR by miR-99a induces autophagy in bladder cancer cells. CONCLUSIONS This is the first study showed that miR-99a markedly inhibits bladder and prostate cancer cell growth via dual inhibition of mTORC1 and mTORC2. miR-99a treatment also induces autophagy through mTOR inhibition. However, the role of miR-99a induces autophagy remains further investigation. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e432 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Te-Fu Tsai More articles by this author Ji-Fan Lin More articles by this author Yi-Chia Lin More articles by this author Kuang-Yu Chou More articles by this author Hung-En Chen More articles by this author Thomas I.S. Hwang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Carrot (DaucuscarotaL.) heat shock protein 70 gene (DcHsp70) confers tolerance to heat or cold stress in E. coli cells

SummaryA heat shock protein 70 gene (DcHsp70) from carrot (Daucus carota L. ‘Mussangochon’) was isolated using PCR.The gene was 1,959 bp-long and the deduced amino acid sequence suggested that the encoded protein was composed of 652 amino acids (approx. 71 kDa). Sequence alignment revealed that the protein contained an ATPase domain at its N-terminus and a substrate-binding domain at its C-terminus. It also had the characteristic EEVD sequence for cytosolic localisation. Reverse transcription-PCR revealed the absence of DcHsp70 transcripts in non-stressed leaf tissue. However, heat or cold stress induced DcHsp70 gene expression. To examine the function of DcHsp70, the coding region of the gene was introduced into Escherichia coli BL21 (DE3) and the product was expressed following induction with isopropyl _-D-1-thiogalactopyranoside (IPTG). Under normal growth temperatures (37°C), the transformed E. coli cells showed similar growth rates to the vector-only control. However, under heat stress (50°C), the transformed E. coli cells displayed higher viability (35%) compared to the vector-only control (15%). Under cold stress (2°C), the transformed E. coli cells also displayed higher viability (71%) compared to the vector-only control (43%).Transformed E. coli cells had higher concentrations of soluble proteins under either stress condition compared to the vector-only control, suggesting that heterologously-expressed DcHSP70 protein functioned as a molecular chaperone to maintain the solubility of proteins. Adenosine triphosphate (ATP) further enhanced the molecular chaperone function of DcHSP70 under either stress condition. The DcHsp70 gene could be a candidate to produce transgenic crops with enhanced heat/cold stress tolerance.

ZRANB2 localizes to supraspliceosomes and influences the alternative splicing of multiple genes in the transcriptome

Alternative splicing is a major source of protein diversity in humans. The human splicing factor zinc finger, Ran-binding domain containing protein 2 (ZRANB2) is a splicing protein whose specific endogenous targets are unknown. Its upregulation in grade III ovarian serous papillary carcinoma could suggest a role in some cancers. To determine whether ZRANB2 is part of the supraspliceosome, nuclear supernatants from human embryonic kidney 293 cells were prepared and then fractioned on a glycerol gradient, followed by Western blotting. The same was done after treatment with a tyrosine kinase to induce phosphorylation. This showed for the first time that ZRANB2 is part of the supraspliceosome, and that phosphorylation affects its subcellular location. Studies were then performed to understand the splicing targets of ZRANB2 at the whole-transcriptome level. HeLa cells were transfected with a vector containing ZRANB2 or with a vector-only control. RNA was extracted, converted to cDNA and hybridized to Affymetrix GeneChip(®) Human Exon 1.0 ST Arrays. At the FDR ≤1.3 significance level we found that ZRANB2 influenced the alternative splicing of primary transcripts of CENTB1, WDR78, C10orf18, CABP4, SMARCC2, SPATA13, OR4C6, ZNF263, CAPN10, SALL1, ST18 and ZP2. Several of these have been implicated in tumor development. In conclusion ZRANB2 is part of the supraspliceosome and causes differential splicing of numerous primary transcripts, some of which might have a role in cancer.

Downregulation of miR-17~92 Expression Increase Paclitaxel Sensitivity in Human Ovarian Carcinoma SKOV3-TR30 Cells via BIM Instead of PTEN

To better understand the molecular mechanisms of paclitaxel resistance in ovarian carcinoma, we evaluated the expression of miRNAs using miRNA microarray between human ovarian carcinoma SKOV3 cells and paclitaxel resistant SKOV3-TR30 cells. Results showed that 69 miRNAs were upregulated while 102 miRNAs were downregulated in SKOV3-TR30 cells. Using real-time PCR, we further clarified that miR-17~92 was overexpressed in SKOV3-TR30 cells compared with that in SKOV3 cells. We then established stable virally transduced SKOV3-TR30-m-PTIP-Sponge all SKOV3-TR30 cells and its vector-only control SKOV3-TR30-m-PTIP-GFP cells. Real time-PCR revealed that SKOV3-TR30-m-PTIP-Sponge all cells expressed approximately 6.18-fold lower levels of miR-17~92 compared with the control group. Decreased expression of miR-17~92 resulted in cell cycle arrest in the G2/M phase and growth inhibition. After the transduction, the BIM protein level was increased in SKOV3-TR30 cells and luciferase reporter assays revealed that miR-17~92 binds directly to the 3′-UTR of BIM. Results of luciferase reporter assays accompanied with Western Blot showed that although miR-17~92 binds directly to the 3′-UTR of PTEN, the PTEN protein expression level was upregulated slightly while the result is of no statistical significance. Our results showed that miR-17~92 could be a causal factor of the downregulation of BIM in SKOV3-TR30 cells and thus induce the paclitaxel resistance in SKOV3-TR30 cells.

Elucidating the Regulon of Multidrug Resistance Regulator RarA in Klebsiella pneumoniae

RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8ΔrarA/pACrarA-2 (rarA-expressing construct) and Ecl8ΔrarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.

Deregulated Activity of AML1/RUNX1 Cooperates with BCR-ABL to Immortalize Hematopoietic Progenitor Cells and Induces Blast Crisis-Like Disease of Chronic Myelogenous Leukemia in Mice,

Abstract 3749Since the introduction of imatinib treatment for chronic myeloid leukemia (CML), most patients in the chronic phase (CP) achieve long-lasting hematologic remission. However, some patients acquire resistance or intolerance to imatinib, which results in disease progression to blast crisis (BC). Although it is known that BCR-ABL induces genomic instability, which results in leukemia cells creating multiple gene defects, genes inducing the progression to BC in CML are largely unknown. Moreover, mutations in the AML1/RUNX1 gene and overrepresentation of AML1/RUNX1 in BC compared to CP have been revealed. We therefore hypothesized that deregulation of AML1/RUNX1 might be one of the mechanisms for disease progression of CML.We initially sought to establish a culture system in vitro that allows rapid identification of genes possibly contributing to the emergence of BC. Given that cells leading to the origin of BC are known to resemble committed progenitor rather than stem cells, we employed Sca-1(-)Lin(-) progenitors derived from mouse fetal liver for the culture. Thus, cells were retrovirally transduced with BCR-ABL alone or in combination with a gene of interest, and cultured in vitro without cytokines. BCR-ABL alone-transduced cells grew initially, but ceased to do so after a week. In contrast, cells co-transduced with BCR-ABL and either one of NUP98-HOXA9, Hes1 or Bmi1 (genes known to induce BC-like disease in concert with BCR-ABL) kept growing and were immortalized in the cytokine-free culture. Importantly, cells cultivated in vitro caused fetal disease when transplanted into mice. These findings suggest the utility of our in vitro culture system as an assay of the co-operativity of a given gene with BCR-ABL to induce BC.We therefore applied the culture system for the assay of AML1/RUNX1 mutants. We chose four AML1/RUNX1 mutants (K83Q, R139G, R80C, and D171N) for the analysis of their co-operativity with BCR-ABL. Wild-type AML1/RUNX1 and a vector-only control were also included. Results showed that K83Q and R139G mutants co-operated with BCR-ABL to immortalize progenitor cells in cytokine-free culture in vitro. In contrast, R80C and D171N mutants lacked such ability. The cells co-expressing BCR-ABL and either K83Q or R139G mutants cultivated in vitro caused fetal disease in mice following transplantation: all 5 mice transplanted with BCR-ABL/K83Q-transduced cells and 7 of 10 mice transplanted with BCR-ABL/R139G-transduced cells developed lethal hematologic diseases 7–79 days following transplantation. These findings are suggestive of variance among mutants of AML1/RUNX1 regarding the ability to induce BC.Unexpectedly, further analyses revealed that wild-type AML1/RUNX1 was as potent as K83Q and R139G in inducing BC: all 4 mice transplanted with BCR-ABL/wild-type AML1-transduced cells developed fetal disease. Examination of various lengths of C-terminally truncated versions of AML1/RUNX1 for their co-operativity with BCR-ABL revealed that AML1L419X and AML1H377X were able to co-operate with BCR-ABL to immortalize progenitor cells, while AML1I337X and AML1A297X did not. These findings suggest that transcriptional regulatory domains residing at the carboxyl-terminal of AML1/RUNX1 play important roles for wild-type AML1/RUNX1 to induce BC-like disease.Perhaps consistent with the increased expression of AML1/RUNX1 contributing to the occurrence of BC, shRNA-mediates silencing of AML1/RUNX1 inhibited growth of human CML cell lines (K562, MegA2, and MEG01).The overall findings of the present study suggest that (1) all mutants of AML1/RUNX1 do not contribute equally to the induction of BC-like disease, but there is a wide range of variance among mutants regarding their ability to induce BC in collaboration with BCR-ABL, and (2) increased expression of wild-type AML1/RUNX1 can induce BC-like disease in collaboration with BCR-ABL in mice. AML1/RUNX1 mutants tested here are known to dominant-negatively inhibit wild-type AML1/RUNX1 functions, but only two of 4 mutants exhibited BC-inducing activity. The over-expression of wild-type AML1/RUNX1 also exhibited such BC-inducing activity. We are currently conducting a detailed investigation of differences in cell lineage affected and growth properties caused by mutant and wild-type AML1/RUNX1s, which may reveal the dichotomic functions of AML1/RUNX1 in the development of BC. Disclosures:Naoe:Zenyaku-Kogyo: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Chugai Pharma.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding; Otsuka Pharma.: Research Funding.

Abstract 482: The HER-2 erbB receptor is vital for growth of breast cancer cells in bone

Abstract HER-2 is a receptor tyrosine kinase that has been shown to be over expressed in 30% of human primary breast cancers (BrCa). Our animal model data and human bone met data, which we present here, indicates that HER-2 up-regulation is required for breast cancer cells to grow in and remodel bone. However, most bone metastases from BrCa will arise from a primary diagnosis of ER positive/HER-2 negative disease. We found that MCF-7 cells, an ER positive/HER-2 negative breast cancer model, formed tumors in bone that had up-regulated HER-2 protein expression. In order to directly test whether or not HER-2 expression has an affect on bone metastasis, we engineered a knock down of the HER-2 receptor in MCF-7 cells by utilizing a lentivirus plasmid containing the shRNA to the HER-2 receptor. Both the shRNA HER-2 knock-down and the vector-only control expressed the Green Fluorescence Protein (GFP) and puromycin resistance for plasmid selection. Our engineered MCF-7 control cells (shVector alone) or the HER-2 knock-down (shHER-2) were injected into the tibia of SCID mice. We found that both the vector-only and HER-2 knock-down cells grew tumors in mouse tibia. Anti-HER-2 immunohistochemical (IHC) analysis showed that the HER-2 protein was re-expressed in the knock down cells in tumors formed in tibiae. Additionally, we found that in eight patients with breast cancer bone metastasis, 100% of these cases contained a significant expression of HER-2 positive cells by IHC. As confirmation of our mouse studies, we found that all of the human bone biopsies obtained from ER positive/HER-2 negative disease were up-regulated for HER-2 in the bone microenvironment. Concomitant with our human data, MCF-7 cells required exogenous estrogen to form tumors in mammary fat pads and did not require exogenous estrogen to form tumors in bone. Therefore, we believe that BrCa cells can change, up-regulate protein expression of HER-2, and become estrogen independent in the bone microenvironment. It may be that these women could benefit from intervention with agents against HER-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 482.

Abstract 1873: Delayed progression to hormone-independent prostate cancer through modulation of mTOR by omega-3 fatty acids

Abstract Prostate cancer is the second leading cause of cancer related deaths in men. The primary obstacle in effective treatment of prostate cancer is the progression of the disease from a hormone-dependent to a hormone-independent state. Previous studies indicate that activation of the mammalian Target of Rapamycin (mTOR) signaling pathway may be involved in progression of prostate cancer. Several studies have demonstrated a significant role for mTOR in promoting androgen independence. Recent data indicate that the combination of androgen deprivation with mTOR inhibition provides an additive anti-tumor effect in vivo. mTOR is directly involved in regulating cell survival, growth, proliferation, and differentiation. Many dietary and lifestyle interventions that reduce cancer risk and progression mediate their effects through regulation of mTOR signaling. Epidemiological studies indicate an inverse relation between dietary intake of omega-3 fatty acids and prostate cancer risk. Omega-3 fatty acids have been shown to inhibit prostate cell growth both in vitro and in vivo. Preliminary data from our laboratory demonstrated that treatment of LNCaP prostate cancer cells with omega-3 fatty acids, specifically docosahexaonoic acid (DHA), delayed development of androgen independence in vitro. This delay correlated with a decrease in protein expression of downstream targets of mTOR regulation, including phosphorylated S6, Bcl-2, and cyclin D. We hypothesize that omega-3 fatty acids may prevent prostate cancer progression to a hormone independent phenotype in part through modulation of mTOR. To test this hypothesis, we transiently transfected LNCaP cells to express a constitutively active mTOR. Our results showed that expression of the constitutively active mTOR correlated with a reduction in response to hormone deprivation, resembling that observed in our previously generated hormone refractory LNCaP cell line. When exposed to hormone-ablation conditions, the proliferation rates observed compared to the that of the vector-only control cells were 64% higher in the constitutively active mTOR cells, and 55% higher in the hormone-refractory cell. Ongoing studies are using a stable transfectant of constitutively active mTOR to determine if modulation of mTOR signaling is one mechanism by which the omega-3 fatty acids delay the progression to hormone-independence. These studies suggest that inhibition of mTOR is critical for cellular response to hormone ablation, and that mTOR may be a viable target for delaying progression to a hormone refractory disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1873.

Involvement of a Broccoli COQ5 Methyltransferase in the Production of Volatile Selenium Compounds

Selenium (Se) is an essential micronutrient for animals and humans but becomes toxic at high dosage. Biologically based Se volatilization, which converts Se into volatile compounds, provides an important means for cleanup of Se-polluted environments. To identify novel genes whose products are involved in Se volatilization from plants, a broccoli (Brassica oleracea var italica) cDNA encoding COQ5 methyltransferase (BoCOQ5-2) in the ubiquinone biosynthetic pathway was isolated. Its function was authenticated by complementing a yeast coq5 mutant and by detecting increased cellular ubiquinone levels in the BoCOQ5-2-transformed bacteria. BoCOQ5-2 was found to promote Se volatilization in both bacteria and transgenic Arabidopsis (Arabidopsis thaliana) plants. Bacteria expressing BoCOQ5-2 produced an over 160-fold increase in volatile Se compounds when they were exposed to selenate. Consequently, the BoCOQ5-2-transformed bacteria had dramatically enhanced tolerance to selenate and a reduced level of Se accumulation. Transgenic Arabidopsis expressing BoCOQ5-2 volatilized three times more Se than the vector-only control plants when treated with selenite and exhibited an increased tolerance to Se. In addition, the BoCOQ5-2 transgenic plants suppressed the generation of reactive oxygen species induced by selenite. BoCOQ5-2 represents, to our knowledge, the first plant enzyme that is not known to be directly involved in sulfur/Se metabolism yet was found to mediate Se volatilization. This discovery opens up new prospects regarding our understanding of the complete metabolism of Se and may lead to ways to modify Se-accumulator plants with increased efficiency for phytoremediation of Se-contaminated environments.

Short-Chain Chromate Ion Transporter Proteins from Bacillus subtilis Confer Chromate Resistance in Escherichia coli

Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily (ywrB and ywrA) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C, respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli, suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm.

Plant Geminivirus Rep Protein Induces Rereplication in Fission Yeast

The replication-associated protein (Rep) of geminiviruses, single-stranded DNA viruses of higher plants, is essential for virus replication. Since these viruses do not encode their own polymerases, Rep induces differentiated plant cells to reenter the cell cycle by interacting with the plant homologues of retinoblastoma proteins in order to activate the host DNA synthesis machinery. We have used fission yeast (Schizosaccharomyces pombe) as a model organism to analyze the impact of ectopically expressed African cassava mosaic virus Rep protein on the cell division cycle in closer detail. Upon expression, Rep showed its characteristic DNA cleavage activity, and about 10% of the cells exhibited morphological changes. They were elongated threefold, on average, and possessed a single but enlarged and less compact nucleus in comparison to noninduced or vector-only control cells. Flow cytometry of Rep-expressing cultures revealed a distinct subpopulation of Rep protein-containing cells with aberrant morphology. The other 90% of the cells were indistinguishable from control cells, and no Rep was detectable. Rep-expressing cells exhibited DNA contents beyond 2C, indicating ongoing replication without intervening mitosis. Because a second open reading frame (ORF), AC4, is present within the Rep gene, the role of AC4 was examined by destroying its start codon within the AC1 ORF. The results confirmed that Rep is necessary and sufficient to induce rereplication in fission yeast. The unique potential of this well-investigated model for dissecting the cell cycle control by geminiviral proteins is discussed.

The Anaphase Promoting Complex Induces Substrate Degradation during Neuronal Differentiation

The anaphase promoting complex (APC) is an E3 ubiquitin ligase required for the metaphase-to-anaphase transition and mitotic exit. However, APC also plays roles in G(1), where it is regulated by Cdh1, and APC activity has also been detected in differentiated and non-proliferating cells, suggesting that it may play roles outside the cell cycle. Here, we report that disrupting APC(Cdh1) activity inhibits neurite outgrowth of both PC12 pheochromocytoma cells and primary cerebellar granule cells. APC(Cdh1) activity dramatically increases as PC12 cells differentiate in response to nerve growth factor. Furthermore, a key target degraded by APC(Cdh1) following nerve growth factor treatment is the F-box protein Skp2, and APC(Cdh1)-mediated destruction of Skp2 is essential for proper terminal differentiation of neuronal precursors.

Activation of the Small GTPase Rac2 via the B Cell Receptor Regulates B Cell Adhesion and Immunological-Synapse Formation

The integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.