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P Ao

Publications and source records attributed to P Ao.

At least 19 recordsLinked to original sources
PubMed2001

Abbreviated cell cycle progression induced by the serine/threonine protein phosphatase inhibitor okadaic acid at concentrations that promote neoplastic transformation.

We examined cell cycle-related effects of the phosphatase inhibitor okadaic acid (OA) in T51B rat liver epithelial cells under conditions chosen to mimic early stages of tumor promotion by this compound. Optimal transformation (colony formation in soft agar) was seen after prolonged culture of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated T51B cells in 7 nM OA. Paradoxically, T51B cells treated with 2-10 nM OA showed decreased, rather than increased, proliferation in response to epidermal growth factor (EGF), as measured by [3H]thymidine incorporation. Complete inhibition was observed within 24 h at 10 nM OA. This response paralleled a loss of EGF-stimulated cdk2 kinase activity and an increase in association of the inhibitors p21 (cip-1) and p27 (kip-1) with cdk2. An increase in p53 phosphorylated on serine 15 accompanied the rise in p21 (cip-1). Both phosphorylation of the retinoblastoma protein and induction of cyclin A by EGF were blocked in cells treated with OA, but there was an increase in cyclin E. Resting cells treated with OA alone also showed elevated cyclin E levels, together with reduced levels of the E2F regulator pRb2/p130. Taken together, these observations indicate transforming levels of okadaic acid elicit a G(1)-trapping effect by facilitating cell cycle progression to the G(1)/S checkpoint, where cells are trapped by mechanisms that include p21 (cip-1)-mediated inhibition of cdk2. They support the premise that disruption of cellular processes regulating the transitions from G(0) to G(1) to S-phase is an important early step in tumor promotion by low levels of okadaic acid.

Animals↗
PubMed2000

Steering an eigenstate to a destination

The control of time evolution of a quantum state under various physical constraints is investigated and solved in the context of a two-level system. We have discovered a general scheme of steering an eigenenergy state to a destination without net nonadiabatic transitions, and we discuss how the result may be tested and utilized in practice.

Journal Article↗
PubMed1999

Disruption of gap junctional communication by the platelet-derived growth factor is mediated via multiple signaling pathways.

The platelet-derived growth factor (PDGF) mediates its cellular functions via activation of its receptor tyrosine kinase followed by the recruitment and activation of several signaling molecules. These signaling molecules then initiate specific signaling cascades, finally resulting in distinct physiological effects. To delineate the PDGF signaling pathway responsible for the disruption of gap junctional communication (GJC), wild-type PDGF receptor beta (PDGFRbeta) and a series of PDGFRbeta mutants were expressed in T51B rat liver epithelial cells. In cells expressing wild-type PDGFRbeta, PDGF induced disruption of GJC and phosphorylation of a gap junctional protein, connexin-43 (Cx43), which required activation of mitogen-activated protein kinase, although involvement of additional factors was also evident. In the F5 mutant lacking binding sites for phosphatidylinositol 3-kinase, GTPase-activating protein, SHP-2, and phospholipase Cgamma1 (PLCgamma1), PDGF induced mitogen-activated protein kinase, but failed to affect GJC or Cx43, indicating involvement of additional signals presumably initiated by one or more of the mutated binding sites. Examination of the single-site mutants revealed that PDGF effects were not mediated via a single signaling component. This was confirmed by the "add-back" mutants, which showed that restoration of either SHP-2 or PLCgamma1 binding was sufficient to propagate the GJC inhibitory actions of PDGF. Further analysis showed that activation of PLCgamma1 is involved in Cx43 phosphorylation, which surprisingly failed to correlate with GJC blockade. The results of our study demonstrate that PDGF-induced disruption of GJC can be mediated by multiple signaling pathways and requires participation of multiple components.

Animals↗
PubMed1999

Impaired expression and posttranslational processing of connexin43 and downregulation of gap junctional communication in neoplastic human prostate cells.

BACKGROUND: Gap junctional communication (GJC) has been implicated in the control of cell proliferation. Numerous cancer cells show a decrease or loss of GJC compared to their normal counterparts. Lack of adequate information on the status of gap junctions during prostate neoplasia prompted us to examine this form of cancer, which comprises about 14% of male cancer deaths in America. METHODS: Cultured normal human prostate epithelial cells and several different human prostate tumor lines were used in this study. GJC was assayed by dye transfer, whereas Western blot and immunofluorescence methods were used to examine connexin43 (Cx43) levels and the presence of gap junctions, respectively. RESULTS: Normal human prostate cultures exhibited extensive cell-communication which was completely absent in all the examined tumor cells. This disrupted communication was associated with a decreased expression and an impaired posttranslational modification of Cx43 in these cells. Abundant immunostaining of gap junctional channels by a Cx43-antibody was observed in normal prostate cells but not in tumor cells. CONCLUSIONS: Our data provide further support for the hypothesis that loss of junctional communication is a critical step in progression to human prostate neoplasia.

Blotting, Western↗
PubMed1999

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate.

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet-derived growth factor (PDGF)-induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor beta (PDGFr beta). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF- or TPA-induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF- and TPA-induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2-mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N-acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC.

Animals↗
PubMed1998

Rapid disruption of gap junctional communication and phosphorylation of connexin43 by platelet-derived growth factor in T51B rat liver epithelial cells expressing platelet-derived growth factor receptor.

Gap junctional communication (GJC) between contacting cells has been postulated to be involved in the regulation of cell proliferation. This suggestion stems from numerous studies showing modulation of GJC by agents that influence cellular proliferation. Platelet-derived growth factor (PDGF), a strong mitogen, inhibits GJC in many cell types. To understand the molecular nature of the signal transduction pathway responsible for the GJC blockade, T51B rat liver epithelial cells, which lack endogenous PDGF receptor (PDGFr), were infected with a retrovirus containing either wild-type full-length cDNA of human PDGFr beta (Kin+) or a mutant PDGFr beta lacking receptor tyrosine kinase activity (Kin-). PDGF caused a complete but transient interruption of cell communication in Kin+ cells within 15-20 min of addition. This interruption of GJC was not associated with a gross destabilization of gap junction plaques but with the phosphorylation of connexin43 (Cx43), the only known gap junction protein expressed in these cells. These effects were exhibited in either control T51B cells or in Kin- cells, indicating a requirement of the receptor tyrosine kinase activity. Further examination revealed that the newly phosphorylated Cx43 then undergoes a rapid degradation utilizing the lysosomal pathway resulting in a decreased total Cx43 protein level. The re-establishment of GJC following PDGF treatment was dependent on protein synthesis. This report describes a suitable cell system which is currently being utilized for the characterization of the PDGF signaling pathway responsible for the inhibition of GJC.

Animals↗
PubMed1998

Platelet-derived growth factor-induced disruption of gap junctional communication and phosphorylation of connexin43 involves protein kinase C and mitogen-activated protein kinase.

Previously we showed a rapid and transient inhibition of gap junctional communication (GJC) by platelet-derived growth factor (PDGF) in T51B rat liver epithelial cells expressing wild-type platelet-derived growth factor beta receptors (PDGFrbeta). This action of PDGF correlated with the hyperphosphorylation of the gap junction protein connexin43 (Cx43) and required PDGFrbeta tyrosine kinase activity, suggesting the participation of protein kinases and phosphatases many of which are activated by PDGF treatment. In the present study, two such kinases, namely protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), are investigated for their possible involvement in PDGF-induced closure of junctional channels and Cx43-phosphorylation. Down-regulation of PKC-isoforms by 12-O-tetradecanoylphorbol-13-acetate or pretreatment with the PKC inhibitor calphostin C, completely blocked PDGF action on GJC and Cx43. Activation of MAPK correlated with PDGF-induced Cx43 phosphorylation, and prevention of MAPK activation by PD98059 eliminated the PDGF effects. Interestingly, elimination of GJC recovery by cycloheximide was associated with a sustained activated-MAPK level. Based on these results we postulate that the activation of PKC and MAPK are required in PDGF-mediated Cx43 phosphorylation and junctional closure.

Animals↗
PubMed1995

Connexin43 reverses the phenotype of transformed cells and alters their expression of cyclin/cyclin-dependent kinases.

Communication between adjacent cells through gap junctions is believed to be involved in the regulation of cell proliferation. This stems in part from the observation that transfection and overexpression of connexin (cx) 32 or cx43 genes into neoplastic cells lead to normalization of growth and decrease their tumorigenicity. The molecular mechanism(s) responsible for this phenomenon has not been characterized. We transfected the rat cx43 gene into a phenotypically transformed dog kidney epithelial cell line, TRMP, and were successful in restoring gap junctional communication as measured by dye coupling. In addition, cx43-transfected clones reverted to a flat morphology and were sensitive to density-dependent inhibition of proliferation with their G1 and S phase duration almost doubled. These cx43-induced effects were coupled with a decreased expression of specific cell cycle regulatory genes critical to cell cycle progression in nonneoplastic cells including cyclin A, D1, D2, and the cyclin-dependent kinases (CDK) 5 and CDK6. The protein levels of cyclin E, CDK2, and CDK4 were not affected. These results suggest that overexpression of cx43 and the formation of gap junctions with the establishment of gap junctional communication can affect the phenotype of transformed cells and alter specific gene expressions involved in cell cycle regulation.

Animals↗
PubMed1995

Inhibition of intimal hyperplasia and occlusion in Dacron graft with heparin and low molecular weight heparin.

OBJECTIVE: Intimal hyperplasia (IH) is a significant cause of late prosthetic graft occlusion. The influence of heparin and low molecular weight heparin (LMWH) on IH was studied in a sheep model. EXPERIMENTAL DESIGN: A gelatin sealed Dacron (Gelsoft) interposition vascular graft was placed end to end in sheep carotid artery. Standard heparin (SH) was administered in a dose of 7,500 units twice daily subcutaneously (n = 6). LMWH (enoxaparin) was administered in two dosages, a "low dose" of 2 mg/kg (n = 6) and a "high dose" of 5 mg/kg (n = 6) subcutaneously daily. A control group received a graft but no heparin (n = 6). At one month the animals were sacrificed and the grafts sectioned. An intimal measurement was obtained under light microscopy using an eyepiece graticule from a longitudinally sectioned graft. RESULTS: Controls occluded five grafts of six and recorded a mean intimal thickness of 2,510 micrometres, SH sheep occluded two with a mean thickness of 83 (p < 0.05), low dose enoxaparin sheep had no occlusions with a mean thickness of 73 (p < 0.001) and high dose enoxaparin sheep occluded one with a mean thickness of 225 (p < 0.01). The reduced occlusion rates were significant for the LMWH groups (p = 0.007 for low dose, p = 0.04 for high dose) but not for SH. There were no significant differences between treatment groups with respect to IH. CONCLUSIONS: In this sheep animal study, each of SH and two LMWH dose regimens resulted in a reduction in Dacron vascular graft occlusion rate and the development of IH, with the greatest effect seen in the low dose LMWH group.

Animals↗