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Involvement of isoprenyl pathway in b-FGF- and EGF-induced proliferation of human prostate organotypic cultures
I N V O L V E M E N T O F I S O P R E N Y L P A T H W A Y IN b FGF- AND EGF-INDUCED PROLIFERATION OF HUMAN PROSTATE ORGANOTYPIC CULTURES. S~V" P A U B E R T - B R A O U E T , D.H. JANSSEN,* N. E N T , A. S E R I K O F F , N DHERSIN, R. D E DECKER*, C. SCHULMAN,* R. K I S S * Bio-Inova P,es LabS, Plaisir France, *ULB, Brussels, Belgium.
ISOPENTENYLADENOSINE INVOLVEMENT IN P2-MEDIATED PROLIFERATION OF CULTURED RAT ASTROCYTES Ciccarelli R., Di Iorio P., Ba!!erini P., D'Alimonte I., Giuliani P., Renzetti A. and Caciagli F. htst.of Pharmacology, University'of Chieti, Medical School, Chieti, Italy. Farncsyl or geranylgeranyl moieties, derived from the cholesterol biosynthesis, modify some cell proteins and facilitate their biological activity on celt growth and receptgr-mediated signal transduction. Less known is the functional role of isopentenyladcnosine (i6-ADO), a modified nucleoside derived from mevalonic acid (MVA) metabolism. Adenosine (ADO) was found to be involved in the control of cell proliferation and we reported that ADO released from rat astrocytes or derived from the breakdown of released ATP inhibits, via A~ receptors, cell proliferation. Conversely, not degradable analogues of ATP cansed a P2-mediatcd mitogenic effect, related to intracellular Ca-~ mobilization and protein kinase C activation, ht this study, primary cultures of rat astrocytes were used to evaluate ~hcther the Pzmediated activation of celt proliferation was associated to i6-ADO formation, lsoprenylated proteins were radiolabelled by culture incubation with an isoprenoid precursor, [3HJ-MVA~ and resolved by gel electrophoresis. [~4C]-ADO was also added to cultnre medium in order to individuate the MVA-derived nucleoside. Tritimn was predominantly incorporated into 21-28 KDa and 65-72 KDa proiein clusters= while the [~4C]-derivatives were selectively present into the clusler of 21-28 KDa proteins. The radioactivity was reduced by 50% in quiescent cells cultured in the absence of sentm, whereas it was enhanced 2.7-fold in cultures stimulated with 100 p,M adenosine-~yimidotriphosphate (AMP-PNP) for 8h. Simvastatiu inhibited in dose-dependent manner (ECho=50 ~M) the AMPPNP-iuduced astroeyte proliferation and strongly reduced the radioactivity in the above mentioned protein cluslers. These effects were reversed by coincubation with MVA. The culture pretreatntent with nitrobcuzyl,6thioinosine (NBTI), an inhibitor of ADO uptake, attenuated the mitogenic effect of AMP-PNP, suppressed the [~4C]-ADO-derivativesin the cluster of 21-28 KDa proteins and reduced the presence of [3HI-labelledproteins also in the cluster of 65-72 KDa. These results suggest that the presence of [3HIand I~'~C]-compounds in the 2 !-28 KDa proteins correlates with the AMPPNP-induced proliferation. Wc speculate that these proteins contains i6ADO derivatives, which re!eased and rc-uptaken ADO is involved in.
Growth factors (mainly D-FGF) may play an important role in the development of benign prostatic hypertrophy (BPH). To assess the involvement of isoprenyt pathway in human prostate cell lines, crganotypic cell cultures of prostate were performed: biopsies minced in small fragments, were placed in Petri dishes containing MEM without other additive. Biopsies were put in culture at tO and b-FGF or EGF (0.5 ng/ml of each) were added. Lovastatin (5 mM) was added at tO + 3 0 rain. Farnesol or geraniol (10.5 M) was added at tO + 24 h. Cultures were stopped at tO + 72 h. [3H] thymidine was added at tO + 36, tO + 48, tO + 60 or tO + 71 h. Fragments were fixed, dehydrated then coated with paraffin and cut in pieces of 5 mm thickness. Histological slides were soaked in a nuclear emulsion, dried, then placed at 4°C for 2 to 3 weeks, revealed, fixed and stained by the hemalun dye. b-FGF significantly increased human prostate cell proliferation (by 100% and 250%); incorporation of [3H] thymidine was mainly affected in glandular epithelium. Only few labeling has been recorded in the other parts of prostate biopsies. Similar results were obtained with EGF. Lovastatin abolished both basal and growth factorstimulated proliferation of human prostate epithelium (EGF by 80-95%; b-FGF by 40-90%; in the majority of biopsies p<0,001 vs basal cell growth). Both farnesol and geraniol, precursors of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, increased cell proliferation and potentiated b-FGF-induced proliferation. The present data suggest that isoprenyl pathway is involved in b-FGF- and EGFinduced proliferation in human prostate epithelium and inhibition of isoprenylation may represent a new approach for the treatment of human BPH.
RECEPTORS AND PROTEIN KINASE C REGULATE CONSTITUTIVE MEMBRANE TRAFFICFROMTHE TRANSGOLGITO THE CELLSURFACE -
Phosph01ipase C Signaling and Cell Growth : Analysis .by Stable Expression of Antisense RNA Nebigi], C, G4
Buccione R,, *Santone L, *Baldassarre M., Di Tullio G., Luini A., and *De Matteis M.A. Laboratory of Molecular Neurobiolegy and *Unit of Phyaiopathology of Secretion, Consorzio Marie Negri Sud, 66030 S, Maria Imbaro (Chieti) Italy.
Moxham_ C, M., Morris A. J., and Malbon, C, C, Molecular Pharmacology-l-lSC, Univer. Med}cal Center. SUNY/ ~mnv Brook. Stony Broor~ NY 11794-8651. USA l ne role or pnuaphoinositide-specific phospholipase C
We have previously shown (Nature, 364:818, 1993) that the binding of ARF and ~-COP to the Golgi complex in rive is stimulated by activation of the IgE receptor in RBL cells through the stimulation of protein kinase C (PKC) and in vitro by PKC activation with phorbol esters. Here we show that PKC activation accelerates constitutive transport from the trans-Golgi/trans-Golgi network (TG/TGN) to the plasma membrane (RM) of both a fluid phase bulk-flow marker, glycosaminoglycan (GAG) chains, and a membrane-bound protein, the vesicular stcmatitis virus G-protein (VSV-G). RBL cells released 2 to 3-fold more GAG as compared to the'control when stimulated with !qhorbol myristate acetate (PMA). BFA inhibited both basal and stimulated release. Furthermore, the rate of appearance of VSV-G on the PM of VSV-infected RBL cells was increased by PMA. PMA effects Were due to PKC activation since a) the stimulation by PMA was completely inhibitable by the highly specific PKC inhibitors Ro31-8220 and Calphostin C; b) inactive phorbols failed to exert any stimulatory effect. PKC-regulated TG/TGN to PM transport is not a feature unique to RBL cells, We have studied and partially characterized this aspect in different cell lines. In all cases PMA increased the rate of release albeit,with different kinetics and effectiveness depending on the cell type. Activation of the FccRI (IgE receptor) in the presence of external calcium induces massive release from secretory granules and acceleration of constitutive traffic in RBL cells. We show that, in the absence of external calcium, Fc~RI activation induces the acceleration of constitutive, but not regulated, secretion as measured by GAG release. We have partially characterized the signalling pathways activated by FcERI and mediating the stimulation of constitutive release. By using the highty specific inhibitors of PKC, Ro31-8220 and of PI3 kinase, Wortmannin, we were able to determine that both PKC and PI3 kinase activities were required for the acceleration of constitutive membrane traffic induced by gc~RI activation. Similarly. we have observed that in the neuroeedocrine cell line PC12, EGF receptor activation potently accelerates constitutive transport. We are currently studying the signalling pathways involved in EGF-mediated regulation of transport in PC12 cells.
growth is not understood. Degenerate-antisease RNA target sequences were tested for their abilities to suppress selectively the expression of specific isoe•zymes o f the PLC-[3. -,/and -~ families, Rat FYO-2B ~epatoma celh expressing a plasmid (p-PEPCK.PLC) harboring anlisense or sense RNA sequences of PLC within the first exon of the inducible liver-specific-PEPCK gene demonstrated sele~ive suppression of each isozyme of the PLC family (PLC-I31, -yI and -6) by its cognate antisense RNA. Loss of PLC signaling (inositot phosphates anti diacylglycerol formation) correlated with the antisease-p-PEPCK-PLC expression in the FTO-2B cell clones. Suppression of specific PLC expression in FTO-2B cells altered cell growth profoundly. The doubling time of cell clones was increased by expression of antisense, but not sensvp-PEPCK-PLC. The rate of cell growth by clones transfected with antisense-p-PEPCK-PLC correlates closely with PLC expression, The cell clones expressing antisense-p-PEPCK-PLC¢ dLsplay the most marked reduction in the rate of growth. The biochemical basis _for the antisense KNA-induceM inhibition of growth appears to be a disruption o f a subset of cytoplasmic signal transduction
Acknowledgments.Thiswork was partially supportedby Ihe Agenzia per la Promozionee Io Sviluppodel Mezzogiomo(PR-2)and by the CNR(ConvenzioneCNR-Mar[oNegriSud).
pathways (ras, mitogen-activated protein kinase, and protein kinase C) regulated by PLC. This strategy permits analysis of PLC
(PLC) in controlling patterns of gnne expression during ceil
function in
developmentin
vit~'o and in vivo. This research is
Supported by the American Cancer Society.
- - 177 - -
ISOPENTENYLADENOSINE INVOLVEMENT IN P2-MEDIATED PROLIFERATION OF CULTURED RAT ASTROCYTES Ciccarelli R., Di Iorio P., Ba!!erini P., D'Alimonte I., Giuliani P., Renzetti A. and Caciagli F. htst.of Pharmacology, University'of Chieti, Medical School, Chieti, Italy. Farncsyl or geranylgeranyl moieties, derived from the cholesterol biosynthesis, modify some cell proteins and facilitate their biological activity on celt growth and receptgr-mediated signal transduction. Less known is the functional role of isopentenyladcnosine (i6-ADO), a modified nucleoside derived from mevalonic acid (MVA) metabolism. Adenosine (ADO) was found to be involved in the control of cell proliferation and we reported that ADO released from rat astrocytes or derived from the breakdown of released ATP inhibits, via A~ receptors, cell proliferation. Conversely, not degradable analogues of ATP cansed a P2-mediatcd mitogenic effect, related to intracellular Ca-~ mobilization and protein kinase C activation, ht this study, primary cultures of rat astrocytes were used to evaluate ~hcther the Pzmediated activation of celt proliferation was associated to i6-ADO formation, lsoprenylated proteins were radiolabelled by culture incubation with an isoprenoid precursor, [3HJ-MVA~ and resolved by gel electrophoresis. [~4C]-ADO was also added to cultnre medium in order to individuate the MVA-derived nucleoside. Tritimn was predominantly incorporated into 21-28 KDa and 65-72 KDa proiein clusters= while the [~4C]-derivatives were selectively present into the clusler of 21-28 KDa proteins. The radioactivity was reduced by 50% in quiescent cells cultured in the absence of sentm, whereas it was enhanced 2.7-fold in cultures stimulated with 100 p,M adenosine-~yimidotriphosphate (AMP-PNP) for 8h. Simvastatiu inhibited in dose-dependent manner (ECho=50 ~M) the AMPPNP-iuduced astroeyte proliferation and strongly reduced the radioactivity in the above mentioned protein cluslers. These effects were reversed by coincubation with MVA. The culture pretreatntent with nitrobcuzyl,6thioinosine (NBTI), an inhibitor of ADO uptake, attenuated the mitogenic effect of AMP-PNP, suppressed the [~4C]-ADO-derivativesin the cluster of 21-28 KDa proteins and reduced the presence of [3HI-labelledproteins also in the cluster of 65-72 KDa. These results suggest that the presence of [3HIand I~'~C]-compounds in the 2 !-28 KDa proteins correlates with the AMPPNP-induced proliferation. Wc speculate that these proteins contains i6ADO derivatives, which re!eased and rc-uptaken ADO is involved in.
Growth factors (mainly D-FGF) may play an important role in the development of benign prostatic hypertrophy (BPH). To assess the involvement of isoprenyt pathway in human prostate cell lines, crganotypic cell cultures of prostate were performed: biopsies minced in small fragments, were placed in Petri dishes containing MEM without other additive. Biopsies were put in culture at tO and b-FGF or EGF (0.5 ng/ml of each) were added. Lovastatin (5 mM) was added at tO + 3 0 rain. Farnesol or geraniol (10.5 M) was added at tO + 24 h. Cultures were stopped at tO + 72 h. [3H] thymidine was added at tO + 36, tO + 48, tO + 60 or tO + 71 h. Fragments were fixed, dehydrated then coated with paraffin and cut in pieces of 5 mm thickness. Histological slides were soaked in a nuclear emulsion, dried, then placed at 4°C for 2 to 3 weeks, revealed, fixed and stained by the hemalun dye. b-FGF significantly increased human prostate cell proliferation (by 100% and 250%); incorporation of [3H] thymidine was mainly affected in glandular epithelium. Only few labeling has been recorded in the other parts of prostate biopsies. Similar results were obtained with EGF. Lovastatin abolished both basal and growth factorstimulated proliferation of human prostate epithelium (EGF by 80-95%; b-FGF by 40-90%; in the majority of biopsies p<0,001 vs basal cell growth). Both farnesol and geraniol, precursors of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, increased cell proliferation and potentiated b-FGF-induced proliferation. The present data suggest that isoprenyl pathway is involved in b-FGF- and EGFinduced proliferation in human prostate epithelium and inhibition of isoprenylation may represent a new approach for the treatment of human BPH.
RECEPTORS AND PROTEIN KINASE C REGULATE CONSTITUTIVE MEMBRANE TRAFFICFROMTHE TRANSGOLGITO THE CELLSURFACE -
Phosph01ipase C Signaling and Cell Growth : Analysis .by Stable Expression of Antisense RNA Nebigi], C, G4
Buccione R,, *Santone L, *Baldassarre M., Di Tullio G., Luini A., and *De Matteis M.A. Laboratory of Molecular Neurobiolegy and *Unit of Phyaiopathology of Secretion, Consorzio Marie Negri Sud, 66030 S, Maria Imbaro (Chieti) Italy.
Moxham_ C, M., Morris A. J., and Malbon, C, C, Molecular Pharmacology-l-lSC, Univer. Med}cal Center. SUNY/ ~mnv Brook. Stony Broor~ NY 11794-8651. USA l ne role or pnuaphoinositide-specific phospholipase C
We have previously shown (Nature, 364:818, 1993) that the binding of ARF and ~-COP to the Golgi complex in rive is stimulated by activation of the IgE receptor in RBL cells through the stimulation of protein kinase C (PKC) and in vitro by PKC activation with phorbol esters. Here we show that PKC activation accelerates constitutive transport from the trans-Golgi/trans-Golgi network (TG/TGN) to the plasma membrane (RM) of both a fluid phase bulk-flow marker, glycosaminoglycan (GAG) chains, and a membrane-bound protein, the vesicular stcmatitis virus G-protein (VSV-G). RBL cells released 2 to 3-fold more GAG as compared to the'control when stimulated with !qhorbol myristate acetate (PMA). BFA inhibited both basal and stimulated release. Furthermore, the rate of appearance of VSV-G on the PM of VSV-infected RBL cells was increased by PMA. PMA effects Were due to PKC activation since a) the stimulation by PMA was completely inhibitable by the highly specific PKC inhibitors Ro31-8220 and Calphostin C; b) inactive phorbols failed to exert any stimulatory effect. PKC-regulated TG/TGN to PM transport is not a feature unique to RBL cells, We have studied and partially characterized this aspect in different cell lines. In all cases PMA increased the rate of release albeit,with different kinetics and effectiveness depending on the cell type. Activation of the FccRI (IgE receptor) in the presence of external calcium induces massive release from secretory granules and acceleration of constitutive traffic in RBL cells. We show that, in the absence of external calcium, Fc~RI activation induces the acceleration of constitutive, but not regulated, secretion as measured by GAG release. We have partially characterized the signalling pathways activated by FcERI and mediating the stimulation of constitutive release. By using the highty specific inhibitors of PKC, Ro31-8220 and of PI3 kinase, Wortmannin, we were able to determine that both PKC and PI3 kinase activities were required for the acceleration of constitutive membrane traffic induced by gc~RI activation. Similarly. we have observed that in the neuroeedocrine cell line PC12, EGF receptor activation potently accelerates constitutive transport. We are currently studying the signalling pathways involved in EGF-mediated regulation of transport in PC12 cells.
growth is not understood. Degenerate-antisease RNA target sequences were tested for their abilities to suppress selectively the expression of specific isoe•zymes o f the PLC-[3. -,/and -~ families, Rat FYO-2B ~epatoma celh expressing a plasmid (p-PEPCK.PLC) harboring anlisense or sense RNA sequences of PLC within the first exon of the inducible liver-specific-PEPCK gene demonstrated sele~ive suppression of each isozyme of the PLC family (PLC-I31, -yI and -6) by its cognate antisense RNA. Loss of PLC signaling (inositot phosphates anti diacylglycerol formation) correlated with the antisease-p-PEPCK-PLC expression in the FTO-2B cell clones. Suppression of specific PLC expression in FTO-2B cells altered cell growth profoundly. The doubling time of cell clones was increased by expression of antisense, but not sensvp-PEPCK-PLC. The rate of cell growth by clones transfected with antisense-p-PEPCK-PLC correlates closely with PLC expression, The cell clones expressing antisense-p-PEPCK-PLC¢ dLsplay the most marked reduction in the rate of growth. The biochemical basis _for the antisense KNA-induceM inhibition of growth appears to be a disruption o f a subset of cytoplasmic signal transduction
Acknowledgments.Thiswork was partially supportedby Ihe Agenzia per la Promozionee Io Sviluppodel Mezzogiomo(PR-2)and by the CNR(ConvenzioneCNR-Mar[oNegriSud).
pathways (ras, mitogen-activated protein kinase, and protein kinase C) regulated by PLC. This strategy permits analysis of PLC
(PLC) in controlling patterns of gnne expression during ceil
function in
developmentin
vit~'o and in vivo. This research is
Supported by the American Cancer Society.
- - 177 - -