Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Protein kinase C potentiation of N-methyl-d-aspartate receptor activity is not mediated by phosphorylation of N-methyl-d-aspartate receptor subunits

N-methyl-d-aspartate receptors (NMDARs) are Ca2+-permeable glutamate-gated ion channels whose physiological properties in neurons are modulated by protein kinase C (PKC). The present study was undertaken to determine the role in PKC-induced potentiation of the NR1 and NR2A C-terminal tails, which serve as targets of PKC phosphorylation [Tingley, W. G., Ehlers, M. D., Kameyama, K., Doherty, C., Ptak, J. B., Riley, C. T. & Huganir, R. L. (1997) J. Biol. Chem. 272, 5157–5166]. Serine residue 890 in the C1 cassette is a primary target of PKC phosphorylation and a critical residue in receptor clustering at the membrane. We report herein that the presence of the C1 cassette reduces PKC potentiation and that mutation of Ser-890 significantly restores PKC potentiation. Splicing out or deletion of other C-terminal cassettes singly or in combination had little or no effect on PKC potentiation. Moreover, experiments involving truncation mutants reveal the unexpected finding that NMDARs assembled from subunits lacking all known sites of PKC phosphorylation can show PKC potentiation. These results indicate that PKC-induced potentiation of NMDAR activity does not occur by direct phosphorylation of the receptor protein but rather of associated targeting, anchoring, or signaling protein(s). PKC potentiation of NMDAR function is likely to be an important mode of NMDAR regulation in vivo and may play a role in NMDA-dependent long-term potentiation.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct.

Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSalpha recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by trans-diamminedichloroplatinum(II) at a d(GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSalpha may be involved in the cytotoxic action of this chemotherapeutic agent.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.