Hypoxia and the metabolic phenotype of prostate cancer cells.

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.

Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport.

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.

ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro.

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

Molecular characterization of SerH3, a Tetrahymena thermophila gene encoding a temperature-regulated surface antigen.

The DNA sequences of a cDNA clone and the macronuclear genomic fragment corresponding to the functional copy of the SerH3 surface antigen gene of Tetrahymena thermophila were determined. Primer extension and nuclease protection assays show that the SerH3 transcription unit is 1,425 nucleotides long and contains no introns. The predicted polypeptide encoded by the SerH3 gene has a molecular mass of 44,415 daltons; one-third of its 439 residues are either cysteine, serine, or threonine. The central half of the polypeptide consists of three homologous domains in tandem array; within these domains, the cysteine, proline, and tryptophan residues occur in highly regular patterns.

Reproducible and variable rearrangements of a Tetrahymena thermophila surface protein gene family occur during macronuclear development.

The expression of Tetrahymena surface proteins serotype H3 (SerH3) and serotype T (SerT) is under environmental regulation. SerH3 is expressed when cells are incubated between the temperatures of 20 and 35 degrees C, while SerT is expressed when cells are grown at temperatures above 35 degrees C. Using a SerH3 cDNA clone as a hybridization probe, we determined that (i) the SerH3 gene is a member of a multigene family; (ii) most members of this multigene family are variably rearranged during macronuclear development; and (iii) the gene which produces the SerH3 mRNA is reproducibly rearranged during macronuclear development.

mRNA stability plays a major role in regulating the temperature-specific expression of a Tetrahymena thermophila surface protein.

Synthesis of the serotype H3 (SerH3) surface antigen is temperature dependent and responds within 1 h to a change in incubation conditions (G.A. Bannon, R. Perkins-Dameron, and A. Allen-Nash, Mol. Cell. Biol. 6:3240-3245, 1986). Recently, a Tetrahymena thermophila cDNA clone (pC6; D.W. Martindale and P.J. Bruns, Mol. Cell. Biol. 3:1857-1865, 1983) has been shown to be homologous to a portion of the SerH3 mRNA (F.P. Doerder and R.L. Hallberg, personal communication), and it was shown that the cellular levels of this RNA rapidly decreased when cells were shifted from 30 to 41 degrees C (R.L. Hallberg, K.W. Kraus, and R.C. Findly, Mol. Cell. Biol. 4:2170-2179, 1984). These observations indicate that synthesis of the SerH3 protein is highly regulated in response to temperature and led us to initiate studies to determine the mechanism(s) by which SerH3 gene expression is controlled. Using pC6 as a hybridization probe for the SerH3 mRNA, we have determined that (i) the level of SerH3 protein synthesis is directly correlated with the amount of SerH3 message available for translation; (ii) there is, at most, a twofold difference between the relative transcription rates of SerH3 genes at 30 and 40 degrees C; (iii) the SerH3 mRNA half-life in cells incubated at 30 degrees C is greater than 1 h, whereas the half-life in cells incubated at 40 degrees C is only approximately 3 min. These results demonstrate that Tetrahymena SerH3 surface protein expression is regulated by mRNA abundance. Furthermore, the major mechanism controlling mRNA abundance is a dramatic temperature-dependent change in SerH3 mRNA stability.

Platelet-derived growth factor (PDGF)-AB-mediated phosphorylation of PDGF beta receptors.

Platelet-derived growth factor (PDGF) stimulates the proliferation of Balb/c-3T3 fibroblasts through binding and subsequent activation of PDGF receptors. Activation of the PDGF receptors has been proposed to involve receptor dimerization. PDGF-AB has been shown to bind PDGF alpha and beta receptor subunits to form PDGF alpha beta and alpha alpha receptor dimers. In this paper we demonstrate that, following the down-regulation of PDGF alpha receptors, the binding of PDGF-AB to beta receptors occurred at 37 degrees C but not at 4 degrees C. PDGF-AB stimulated the phosphorylation of PDGF beta receptor monomers in cells depleted of PDGF alpha receptors by prior exposure to PDGF-AA.

GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles.

Upon addition of GTPgammaS 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 alpha1, 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 GTPgammaS 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 GTPgammaS. 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.