RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53؉/؊ matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity ϳ4-to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.
Guanine nucleotide binding protein (Gprotein)-coupled receptor kinases (GRKs) specificaily phosphorylate the agonist-occupied form of G-protein-coupled receptors such as the P2-adrenergic receptor and rhodopsin. The best characterized members of this family include the ,3ad-renergic receptor kinase (.BARK) and rhodopsin kinase. To identify additional members of the GRK family, the polymerase chain reaction was used to amplify human heart cDNA using degenerate oligonucleotide primers from highly conserved regions unique to the GRK family. Here we report the isolation of a cDNA that encodes a 590-amino acid protein kinase, termed GRK5, which has 34.8% and 47.2% amino acid identities with B3ARK and rhodopsin kinase, respectively. Interestingly, GRK5 has an even higher homology with DrosophUa GPRK-2 (71.0% identity) and the recently identified human ff11 (69.1% identity). Northern blot analysis of GRK5 with selected human tissues reveals a message of -3 kilobases with highest levels in heart, placenta, lung > skeletal muscle > brain, liver, pancreas > kidney. GRK5, overexpressed in SM insect cells using the baculovirus system, was able to phosphorylate rhodopsin in a light-dependent manner. In addition, GRK5 neither contains a consensus sequence for isoprenylation like rhodopsin kinase nor is activated by G-protein vysubunits like I3ARK1. Thus, GRK5 represents a member of the GRK family that likely has a unique physiological role.Guanine nucleotide binding protein (G protein)-coupled receptors represent a diverse family of cell-surface proteins that transduce the binding ofextracellular ligands (hormones, neurotransmitters, odorants, light, etc.) into intracellular signaling events (1, 2). Two of the best characterized G-protein-coupled receptors are the hormone-responsive P2-adrenergic receptor (P32AR), which mediates catecholamine stimulation of adenylyl cyclase, and the visual "light receptor" rhodopsin, which mediates phototransduction in retinal rod cells (3, 4). The (2AR and rhodopsin share many structural and functional similarities including a conserved protein topology (e.g., seven transmembrane domains) as well as an ability to specifically interact with G proteins upon activation. The similarities between these receptors also extend to mechanisms involved in receptor regulation (3)(4)(5). In both systems, rapid receptor desensitization or loss ofresponsiveness is promoted by phosphorylation of the receptor. This phosphorylation is mediated by protein kinases that have the unique ability to recognize and phosphorylate their receptor substrates only when they are in their active conformationsi.e., when they have been stimulated and/or occupied by appropriate agonist ligands (6). The fadrenergic receptor kinase (P3ARK) (7,8) and rhodopsin kinase (9, 10) have been identified as the kinases involved in the agonist-specific phosphorylation of the 32AR and rhodopsin, respectively.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby mark...
Prostaglandins are arachidonic acid metabolites that may play a major role as mediators of cellular function. PGF 2␣ 1 has diverse physiological actions ranging from being a potent luteolytic agent (1, 2) to causing smooth muscle contraction in the uterus (3, 4), vasculature (5), and gastrointestinal (6) and respiratory tracts (7,8). PGF 2␣ induces DNA synthesis and cell proliferation in 3T3 fibroblasts (9, 10). Neuronal astrocytes respond to PGF 2␣ , which may mediate pain transmission (11). Recently, PGF 2␣ has also been shown to cause hypertrophy of cardiac myocytes and induction of myofibrillar genes, independent of muscle contraction. These observations suggest a role for the eicosanoid during development, in compensatory hypertrophy and/or in recovery of the heart from injury (12). Recently, PGF 2␣ analogs have been shown to reduce intraocular pressure (IOP), in patients with glaucoma (13,14). Although the precise mechanisms involved remain unclear, the effects of PGF 2␣ analogs on IOP may be attributed, at least in part, to their actions on the ciliary muscle. PGF 2␣ reduces IOP by increasing the uveoscleral outflow of aqueous humor (15, 16), possibly by reducing the resistance between the ciliary muscle bundles, via an effect on the extracellular matrix (17).A single PGF 2␣ receptor (FP) has been cloned from myometrial tissue (18 -22). Given that there is evidence consistent with splice variation of the FP (23), as has been described for other prostanoid receptors (24, 25), we wished to address the possibility that a distinct isoform might mediate the actions of PGF 2␣ in the ciliary muscle. Clarification of the nature of the human ciliary FP and development of an antibody that specifically recognized the receptor protein would facilitate investigation of the effects of PGF 2␣ and its analogs on IOP.PGF 2␣ is formed from arachidonic acid via metabolic transformation sequentially catalyzed by phospholipases, cyclooxygenases, and a specific PGF synthase (26). However, it is now appreciated that a series of PGF 2␣ isomers, the F 2 isoprostanes, may also be formed in vivo via a free radical-dependent pathway (27-29). It has been speculated that these F 2 isoprostanes may function as incidental ligands at eicosanoid receptors, and, possibly, activate related receptors of their own (30). To date, attention has focused particularly on 8-iso-PGF 2␣ . This compound is a potent vasoconstrictor. It is also a mitogen and may activate human platelets (31-33). Curiously, despite its F prostaglandin configuration, 8-iso-PGF 2␣ has been shown to activate thromboxane receptors (TPs), and its biological effects are blocked by TP antagonists (31-33).We now report the cloning of an FP receptor from the human ciliary body (hcb) cDNA library and its localization on the cell membrane. The gene product is identical to that cloned from
Prostaglandin receptors may be activated by their cognate ligand or by free radical catalyzed isoprostanes, products of arachidonic acid peroxidation. For example, prostaglandin F 2␣ (PGF 2␣ ) causes hypertrophy of neonatal rat ventricular myocytes, via the PGF 2␣ receptor (FP). However, the FP may also be activated by the isoprostane, 8,12-iso-iPF 2␣ -III (Kunapuli, P., Lawson, J. A., Rokach, J., and FitzGerald, G. A. (1997) J. Biol. Chem. 272, 27147-27154). Both ligands induce myocyte hypertrophy with overlapping potencies. Interestingly, the hypertrophic effects of these two agonists on cardiomyocytes are additive. Furthermore, the preference of these two agonists for activation of intracellular signal transduction pathways differs in several respects. Thus, PGF 2␣ and 8,12-isoiPF 2␣ -III stimulate inositol phosphate formation with EC 50 values of 50 ؎ 12 nM and 3.5 ؎ 0.6 M, respectively. Moreover, PGF 2␣ causes a robust activation (ϳ50-fold) of Erk2, whereas 8,12-iso-iPF 2␣ -III has no effect. Similarly, PGF 2␣ causes translocation of cytosolic phospholipase A 2 and also results in a 7-fold increment in the formation of 6-keto-PGF 1␣ , whereas 8,12-iso-iPF 2␣ -III exerts no effect on this pathway. On the other hand, both agonists are equally potent in activating JNK1 and c-Jun, whereas neither activates the p38 kinase. Both PGF 2␣ and 8,12-isoiPF 2␣ -III activate the p70S6 kinase (p70 S6K ), but not Akt, downstream of phosphatidylinositol-3-kinase (PI3K). However, both wortmannin, a PI3K inhibitor, and rapamycin, an inhibitor of p70 S6K activity, inhibit 8,12-isoiPF 2␣ -III -induced myocyte hypertrophy, with IC 50 values of 60 ؎ 12 and 3 ؎ 1.7 nM, respectively, whereas neither compound abrogates the PGF 2␣ -mediated response. Thus, both PGF 2␣ and 8,12-iso-iPF 2␣ -III induce myocyte hypertrophy via discrete signaling pathways. Although both agonists signal via the JNK pathway to initiate changes in c-Jun-dependent gene transcription, PGF 2␣ preferentially activates the MEK-Erk2-cytosolic phospholipase A 2 pathway. In contrast, the PI3K-p70 S6K pathway appears to be essential for 8,12-iso-iPF 2␣ -III-induced myocyte hypertrophy. Prostaglandins (PGs)1 are arachidonic acid metabolites that are produced in a wide variety of tissues in response to mechanical and chemical stimuli. The actions of PGF 2␣ are thought to be mediated via the PGF 2␣ receptor (FP), which is a member of the G protein-coupled receptor (GPCR) superfamily (1). PGF 2␣ has diverse physiological actions, ranging from being a potent luteolytic agent (2) to causing vascular smooth muscle contraction (3). In the myocardium, the formation of PGs is induced by pressure overload (4) which can result in cardiac hypertrophy (5, 6). Conversely, PG synthase inhibitors diminish the hypertrophic response induced by hypertension (7).Prostaglandin F 2␣ (PGF 2␣ ) has recently been shown to stimulate hypertrophy of neonatal rat ventricular myocytes and to induce the expression of myofibrillar genes, independent of muscle contraction (8). Paoni and co-workers (...
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