Cre-loxP mediated genoinic targeting to develop rapid and reproducible expression of recombinant proteins in mammalian cells.

PhDExpression levels of transgenes in mammalian cells show extreme variability between individual clones isolated from a single transfection. This is due to differing number of copies integrated into the genome and also from chromosomal "position effects". Therefore extensive screening is required to isolate a suitable cell line for high level expression of a recombinant protein. In this work, the Cre-loxP site-specific recombination system was investigated to eliminate such problems by directing the rapid targeting of any input DNA to a single pre-selected site in Chinese hamster ovary (CHO) cell line. Cre is a 38 kDa recombinase encoded by the bacteriophage P1 which mediates recombination between a pair of specific 34 bp target sequencesc alled loxP sites. The recombination reaction was first investigated in vitro to establish which kinetic parameters could be relevant for efficient gene targeting: a recombinant baculovirus was constructed with a hexa-histidine-cre fusion gene. The activity of Cre protein purified (by single step, hexa-histidine/nickel-bindinga, ffinity chromatography) from infected insect cells was verified by: (i) Cre-loxP interaction in gel retardation assays and (ii) Cre-mediated intramolecular excision between two loxP sites flanking a lacZ gene in a plasmid DNA. To investigate Cre-mediated targeting of an exogenous DNA to a chromosomal loxP site, three CHO cell lines were constructed, each carrying a loxP site between a ß-actin promoter and a secreted alkaline phosphatase (SAP) gene as a reporter. To demonstrate the targeting event, a promoterless lacZ/neor gene was cotransfected with either a cre plasmid or the purified Cre protein from the baculovirus/insect system. Proper targeting should activate expression of f 3- galactosidase from the chromosomal 0-actin promoter and give loss or reduction of SAP expression in G418 resistant transformants. Southern blot analysis showed targeted events mediated by both cre plasmid and recombinant Cre protein. This work should allow the development of a generic mammalian cell line by incorporating the Cre-loxP system for rapid and reproducible large scale production of recombinant proteins.Medical Research Council The Wellcome Foundation Limited post-graduate CASE studentship

Relationship between Tumor DNA Methylation Status and Patient Characteristics in African-American and European-American Women with Breast Cancer

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy naïve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients

Pathway-based expression profiling of benign prostatic hyperplasia and prostate cancer delineates an immunophilin molecule associated with cancer progression

Aberrant restoration of AR activity is linked with prostate tumor growth, therapeutic failures and development of castrate-resistant prostate cancer. Understanding the processes leading to ARreactivation should provide the foundation for novel avenues of drug discovery. A differential gene expression study was conducted using biopsies from CaP and BPH patients to identify the components putatively responsible for reinstating AR activity in CaP. From the set of genes upregulated in CaP, FKBP52, an AR co-chaperone, was selected for further analysis. Expression of FKBP52 was positively correlated with that of c-Myc. The functional cross-talk between c-Myc and FKBP52 was established using c-Myc specific-siRNA to LNCaP cells that resulted in reduction of FKBP52. A non-canonical E-box sequence housing a putative c-Myc binding site was detected on the FKBP4 promoter using in silico search. LNCaP cells transfected with the FKBP52 promoter cloned in pGL3 basic showed increased luciferase activity which declined considerably when the promoter-construct was co-transfected with c-Myc specific-siRNA. ChIP-PCR confirmed the binding of c-Myc with the conserved E-box located in the FKBP52 promoter. c-Myc downregulation concomitantly affected expression of FGF8. Since expression of FGF8 is controlled by AR, our study unveiled a novel functional axis between c-Myc, AR and FGF8 operating through FKBP52

DNA methylation and aberrant expression of Sprouty1 in human prostate cancer

Sprouty1 is a negative regulator of fibroblast growth factor signaling with a potential tumor suppressor function in prostate cancer (PCa). Sprouty1 is downregulated in human PCa and Sprouty1 expression can markedly inhibit PCa proliferation in vitro. The aim of this study was to investigate the role of DNA methylation in Sprouty1 expression in human prostate tumors. We used pyrosequencing to quantitatively measure the methylation status of the Sprouty1 promoter region in prostate tissues and cell lines and assessed Sprouty1 mRNA expression by quantitative RT-PCR. Our data demonstrates significantly higher % methylation of Sprouty1 promoter in the PCa tissues when compared to matched normal tissues. Hypermethylation of Sprouty1 promoter was detected in PCa cell lines compared to the normal prostate epithelial cells. The increased % methylation was associated with reduced Sprouty1 mRNA expression in the PCa tissues and cell lines. Methylation modification of the Sprouty1 promoter using Sss1 methylase abolished promoter activity whereas global demethylation with 5′-Aza-2′-Deoxycytidine treatment induced Sprouty1 expression. Our data demonstrates that DNA methylation in the Sprouty1 promoter region is responsible for downregulating Sprouty1 expression in prostate cancer. © 2009 Landes Bioscience

DNA methylation and exposure to violence among African American young adult males

Exposure to violence (ETV) has been linked to epigenomics mechanisms such as DNA methylation (DNAm). We used epigenetic profiling of blood collected from 32 African American young adult males who lived in Washington DC to determine if changes in DNAm at CpG sites affiliated with nervous and immune system were associated with exposure to violence. Pathway analysis of differentially methylated regions comparing high and low ETV groups revealed an enrichment of gene sets annotated to nervous system and immune ontologies. Many of these genes are known to interact with each other which suggests DNAm alters gene function in the nervous and immune system in response to ETV. Using data from a unique age group, young African American adult males, we provide evidence that lifetime ETV could impact DNA methylation in genes impacted at Central Nervous System and Immune Function sites. Method: Methylation analysis was performed on DNA collected from the blood of participants classified with either high or low lifetime ETV. Illumina®MethylationEPIC Beadchips (~850k CpG sites) were processed on the iScan System to examine whole-genome methylation differences. Differentially methylated CpG-sites between high (n ​= ​19) and low (n ​= ​13) groups were identified using linear regression with violence and substance abuse as model covariates. Gene ontology analysis was used to identify enrichment categories from probes annotated to the nearest gene. Results: A total of 595 probes (279 hypermethylated; 316 hypomethylated) annotated to 383 genes were considered differentially methylated in association with ETV. Males with high ETV showed elevated methylation in several signaling pathways but were most impacted at Central Nervous System and Immune Function affiliated sites. Eight candidate genes were identified that play important biological roles in stress response to violence with HDAC4 (10%), NR4A3 (11%), NR4A2 (12%), DSCAML1(12%), and ELAVL3 (13%) exhibiting higher levels in the low ETV group and DLGAP1 (10%), SHANK2 (10%), and NRG1(11%) having increased methylation in the high ETV group. These findings suggest that individuals subjected to high ETV may be at risk for poor health outcomes that have not been reported previously

Correlating blood-based DNA methylation markers and prostate cancer risk in African-American men.

The objective of this work was to investigate the clinical significance of promoter gene DNA methylation changes in whole blood from African-American (AA) men with prostate cancer (PCa). We used high throughput pyrosequencing analysis to quantify percentage DNA methylation levels in a panel of 8 genes (RARβ2, TIMP3, SPARC, CDH13, HIN1, LINE1, CYB5R2 and DRD2) in blood DNA obtained from PCa and non-cancerous controls cases. Correlations of methylation status and various clinicopathological features were evaluated. Six genes tested achieved significant difference in DNA methylation levels between the PCa compared to control cases (P < 0.05). The TIMP3 loci demonstrated significant correlation of DNA methylation with age for all cases analyzed (p < 0.05). We observed an inverse correlation between CDH13 methylation (p = 0.045; r = -0.21) and serum vitamin D level whereas TIMP3 methylation (p = 0.021; r = -0.24) and DRD2 methylation (p = 0.056; r = -0.201) showed inverse correlation with supplementary vitamin D in the cancer cases. We also observed a direct correlation between methylation of RARβ2 (p = 0.0036; r = 0.293) and SPARC (p = 0.0134; r = 0.20) loci with PSA level in the controls but not the cancer cases. In addition, alcohol cases significantly correlated with higher RARβ2 methylation (p = 0.0314) in comparison with non-alcohol cases. Furthermore, we observed an inverse correlation of DRD2 methylation (p = 0.0349; r = -0.343) and Gleason score. Our data suggests that promoter methylation occurred more frequently in the blood of AA PCa and is associated with various clinicopathological features in AA men with PCa