Recombinant adenoviruses are one of the most common vehicles for efficient in vitro and in vivo gene deliveries. Here, we investigate whether exogenous precursor terminal protein (pTP) expression in 293 cells improves the efficiency of adenovirus packaging and amplification. We used a piggyBac transposon-based vector and engineered a stable 293 line that expresses high level of Ad5 pTP, designated as 293pTP. Using the AdBMP6-GLuc that expresses green fluorescent protein (GFP), BMP6 and Gaussia luciferase, we found that the infectivity of AdBMP6-GLuc viral samples packaged in 293pTP cells was titrated up to 19.3 times higher than that packaged in parental 293 cells. AdBMP6-GLuc viral samples packaged in 293pTP cells exhibited significantly higher transduction efficiency in 143B and immortalized mouse embryonic fibroblast (iMEF) cells, as assessed by fluorescence-activated cell sorting analysis of GFP-positive cells, the luciferase activity assay and BMP6-induced osteogenic marker alkaline phosphatase activities in iMEFs. When adenovirus amplification efficiency was analyzed, we found that 293pTP cells infected with AdBMP6-GLuc yielded up to 12.6 times higher titer than that in parental 293 cells, especially at lower multiplicities of infection. These results strongly suggest that exogenous pTP expression may accelerate the packaging and amplification of recombinant adenoviruses. Thus, the engineered 293pTP cells should be a superior packaging line for efficient adenovirus production.
Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception. This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.
Background: Basal-like breast cancer (BLBC) disproportionally affects younger women and African American women, displaying aggressive clinical behavior with poor outcomes. BLBC is difficult to treat due to its lack of defined molecular targets, and with current treatment, patients often develop metastatic disease. A growing body of evidence points to long noncoding (lnc) RNAs as mediators of tumor progression, suggesting a new class of targets in cancer therapy. While dynamic changes in lncRNA expression are observed across many types of cancer, our understanding of their role in cancer biology is limited. The aim of this study is to identify lncRNAs differentially expressed in BLBC and to characterize the functional features of candidate lncRNA. Methods: Breast tumors and normal breast tissues were recruited from the U of Chicago Breast Cancer Tissue Bank under IRB approved protocols. Microarray profiling was done using Human LncRNA Array v3 (Arraystar) containing 30,586 lncRNAs and 26,109 mRNAs. Non-poly(A) RNAs were included with ribo-zero RNA-seq (Illumina HiSeq 4000). Molecular subtype of breast tumors was determined by PAM50 intrinsic classifier. In addition, CpG methylation of lncRNA promoter regions was analyzed with TCGA HumanMethylation450 Array data from 588 breast tissues. We performed in vitro studies using antisense oligonucleotide (ASO) knockdown and CRISPR-on overexpression in BLBC cell lines. Phenotypic consequences of perturbations of specific lncRNA expression were assessed using proliferation, apoptosis, cell cycle and homologous recombination assays. Results: Microarray profiling of breast tissues from 30 African American women revealed a unique lncRNA signature in basal-like tumors compared to non-basal like tumors. Hundreds of lncRNAs were specifically expressed in BLBC, with >100-fold differences in some lncRNAs compared to tumors from other subtypes or normal breast tissues. Through statistical and in silico analyses we selected lncRNAs displaying increased expression in basal-like tumors (vs non-basal tumors) and BLBC cell lines (vs non-basal cell lines) with a trend of higher expression in cells isolated from women of African ancestry for functional studies. RNA-seq of 50 additional breast tissues recapitulated the distinct lncRNA clustering within the basal-like subtype. TCGA analysis of CpG islands in several lncRNA promoter regions revealed hypo-methylation when compared to non-BLBC, signifying subtype specific epigenetic regulation. ASO knockdown of candidate lncRNAs significantly increased apoptosis and decreased proliferation while CRISPR-on overexpression decreased cell sensitivity to doxorubicin treatment. Depletion of specific lncRNAs caused an increased proportion of cells in G2 phase of the cell cycle, indicating that overexpression of particular lncRNAs may contribute to aberrant cell cycle progression. Cis and trans-regulation of gene expression by lncRNAs is currently under investigation through RNA immunoprecipitation and genome-wide gene expression profiling. Conclusion: We propose that subtype specific lncRNAs function as onco-RNAs specific to BLBC, driving cellular proliferation and increasing resistance to DNA damaging chemotherapies through regulation of the cell cycle. Citation Format: Boatman SM, Han Y-J, Zhang J, Zheng Y, Yeh AC, Olopade OI. Molecular subtype-specific expression of long noncoding RNA regulates proliferation of basal-like breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-08.
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