Upon addition of GTPγS to in vitro budding reactions, COP I vesicles form but retain their coat, making them easy to isolate and analyze. We have developed an in vitro budding assay that reconstitutes the formation of COP I-derived vesicles under conditions where GTP hydrolysis can occur. Once formed, vesicles are uncoated and appear functional as they fuse readily with acceptor membranes. Electron microscopy shows a homogeneous population of uncoated vesicles that contain the medial/trans Golgi enzyme α1,2-mannosidase II. Biochemical quantitation of vesicles reveals that resident Golgi enzymes are up to 10-fold more concentrated than in donor membranes, but vesicles formed in the presence of GTPγS show an average density of resident Golgi enzymes similar to that seen in donor membranes. We show that the sorting process is mediated by the small GTPase arf-1 as addition of a dominant, hydrolysis-deficient arf-1 Q 71 L mutant produced results similar to that of GTPγS. Strikingly, the average density of the anterograde cargo protein, polymeric IgA receptor, in COP I-derived vesicles was similar to that found in starting membranes and was independent of GTP hydrolysis. We conclude that hydrolysis of GTP bound to arf-1 promotes selective segregation and concentration of Golgi resident enzymes into COP I vesicles.

Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis-side and exit in transport vesicles budding from the trans-side. Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretory flow must be recycled constantly by retrograde transport in the opposite direction. In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells. We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cisternal membranes. These vesicles appeared to be depleted of secretory cargo. They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus. Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.

Many cancer cells have an unusual ability to grow in hypoxia, but the origins of this metabolic phenotype remain unclear. We compared the metabolic phenotypes of three common prostate cancer cell models (LNCaP, DU145, PC3), assessing energy metabolism, metabolic gene expression, and the response to various culture contexts (in vitro and xenografts). LNCaP cells had a more oxidative phenotype than PC3 and DU145 cells based upon respiration, lactate production, [ATP], metabolic gene expression, and sensitivity of these parameters to hypoxia. PC3 and DU145 cells possessed similar Complex II and mtDNA levels, but lower Complex III and IV activities, and were unresponsive to dinitrophenol or dichloroacetate, suggesting that their glycolytic phenotype is due to mitochondrial dysfunction rather than regulation. High passage under normoxia converted LNCaP from oxidative to glycolytic cells (based on respiration and lactate production), and altered metabolic gene expression. Though LNCaP-derived cells differed from the parental line in mitochondrial enzyme activities, none differed in mitochondrial content (assessed as cardiolipin levels). When LNCaP-derived cells were grown as xenografts in immunodeficient mice, there were elements of a hypoxic response (e.g., elevated VEGF mRNA) but line-specific changes in expression of select glycolytic, mitochondrial and fatty acid metabolic genes. Low oxygen in vitro did not influence the mRNA levels of SREBP axis, nor did it significantly alter triglyceride production in any of the cell lines suggesting that the pathway of de novo fatty acid synthesis is not directly upregulated by hypoxic conditions. Collectively, these studies demonstrate important differences in the metabolism of these prostate cancer models. Such metabolic differences would have important ramifications for therapeutic strategies involving metabolic targets.

The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G 1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in normal prostate fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1-overexpressing fibroblasts (NPF cyclin D1

Targeting glyoxalases metabolic pathway may represent a strategy to selectively inhibit advanced PCa. Prostate 77:196-210, 2017. © 2016 Wiley Periodicals, Inc.