The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system discovered using bacteria has been repurposed for genome editing in human cells. Transient expression of the editor proteins (e.g. Cas9 protein) is desirable to reduce the risk of mutagenesis from off-target activity. Using the specific interaction between bacteriophage RNA-binding proteins and their RNA aptamers, we developed a system able to package up to 100 copies of Staphylococcus aureus Cas9 ( SaCas9 ) mRNA in each l enti v irus- l ike bionano p article (LVLP). The SaCas9 LVLPs mediated transient SaCas9 expression and achieved highly efficient genome editing in the presence of guide RNA. Lower off-target rates occurred in cells transduced with LVLPs containing SaCas9 mRNA, compared with cells transduced with adeno-associated virus or lentivirus expressing SaCas9 . Our LVLP system may be useful for efficiently delivering Cas9 mRNA to cell lines and primary cells for in vitro and in vivo gene editing applications.

High levels of reactive oxygen species (ROS) present in human prostate epithelia are an important etiologic factor in prostate cancer (CaP) occurrence, recurrence, and progression. Androgen induces ROS production in the prostate by a yet unknown mechanism. Here, to the best of our knowledge, we report for the first time that androgen induces an overexpression of spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in the polyamine oxidation pathway. As prostatic epithelia produce a large excess of polyamines, the androgeninduced polyamine oxidation that produces H 2 O 2 could be a major reason for the high ROS levels in the prostate epithelia. A small molecule polyamine oxidase inhibitor N,N '-butanedienyl butanediamine (MDL 72,527 or CPC-200) effectively blocks androgen-induced ROS production in human CaP cells, as well as significantly delays CaP progression and death in animals developing spontaneous CaP. These data show that polyamine oxidation is not only a major pathway for ROS production in prostate, but inhibiting this pathway also successfully delays CaP progression. [Cancer Res 2009;69(19):7689-95]

Purpose: The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation. Experimental Design: Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells. Results: SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation.We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-nB ligand expression was up-regulated in cells expressing PSA compared with controls. Conclusions: Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin^receptor activator of NF-nB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.Bone is the frequent site of many types of cancer metastasis including prostate cancer. Advanced prostate cancer is frequently accompanied by the development of unique bone metastases characterized as osteoblastic (bone forming), which results in significant complications including bone pain, fractures, and spinal cord compression, even hemiparesis, leading to morbidity with no curable treatment (1 -3). Although osteoblastic lesions are the most dominant bone metastases associated with prostate cancer, osteoclastic lesions (bone resorption) also infrequently occur in prostate bone metastases (4, 5). In contrast, cancers from other tissues that metastasize to bone are frequently associated with osteoclast formation. Osteoblastic characterization associated with prostate bone metastases suggests that factors derived from prostate cancer cells which influence bone remodeling may be unique from other types of cancer cells.Several osteogenic factors produced by prostate cancer cells have been identified including bone morphogenetic proteins (BMP; refs. 6, 7), endothelin-1 (8), insulin-like growth factors (IGF; ref. 9), parathyroid hormone -related peptide (10), transfor...

Based on these data, we conclude that JunD is an essential mediator of the androgen-induced increase in ROS levels in LNCaP cells.