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J C Lacal

Publications and source records attributed to J C Lacal.

At least 37 records · Page 2Linked to original sources
PubMed1998

Wortmannin, an inhibitor of phosphatidyl-inositol 3-kinase, induces oocyte maturation through a MPF-MAPK-dependent pathway.

Wortmannin has been shown to be a non-competitive and irreversible inhibitor of PI3 kinase. For this reason, it has attracted considerable interest and it has been used, as a selective inhibitor of the PI3 kinase, for the study of signal transduction pathways in different systems including Xenopus oocytes. We show here that wortmannin itself is able to induce meiotic maturation at doses slightly higher that those required for complete inhibition of PI3 kinase. This effect was shown to be independent of the ability to inhibit PI3K since another unrelated PI3K inhibitor, LY294002, was unable to induce oocyte maturation at inhibitory concentrations for PI3 kinase. The mechanism for wortmannin-induced maturation involves the activation of maturation promoting factor (MPF) and MAP kinase activities in a time course that preceded the appearance of germinal vesicle breakdown. Thus, the pathway activated by wortmannin directly or indirectly affects other protein or proteins, besides PI3 kinase, responsible for its activity. This new target is placed independently or downstream of the PI3 kinase inhibition and upstream of protein synthesis. Moreover, the inhibition of either MPF or cAMP phosphodiesterase blocks wortmannin-induced maturation. We conclude that wortmannin may be a valuable tool for the study of the pathway leading to mitotic maturation of oocytes, but cannot be used as a specific PI3 kinase inhibitor.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Rho proteins induce metastatic properties in vivo.

Rho proteins have been implicated in the regulation of multiple signal transduction processes. Some of the members of this family, including the rho gene from Aplysia californica and the human genes (rhoA, rhoB and rac-1), are proto-oncogenes since when properly mutated they can induce cell transformation, and the generated rho-transformed cells are tumorigenic when inoculated into mice. In addition to their tumorigenic activity, there is evidence suggesting that Rho proteins may contribute to the metastatic phenotype. However, all the experiments implicating Rho proteins or Rho-regulating proteins in the induction of metastatic potential are either indirect or have been performed in vitro. In this study we investigated whether cells transformed by rho oncogenes do have metastatic potential in vivo. We present evidence that cells transformed by the Aplysia californica rho gene, when injected directly into the blood stream are able to efficiently colonize lungs and secondary organs, consistent with the acquisition of the metastatic potential. Moreover, tumors derived from subcutaneous injections of these rho-transformed cells are also able to metastasize in distant organs, a strong support to the hypothesis that Rho proteins play a role in the metastatic phenotype. Finally, cells transformed by the human oncogenes dbl, vav and ost, three well-known guanine exchange factors for members of the Rho family, or cells transformed by the activated human rac-1 or rhoA genes do also have metastatic potential when injected into the blood stream. These results demonstrate that signaling pathways regulated by Rho proteins play an important role in the acquisition of the metastatic phenotype in vivo.

3T3 Cells↗
PubMed1997

Micro-injection of recombinant lysyl oxidase blocks oncogenic p21-Ha-Ras and progesterone effects on Xenopus laevis oocyte maturation.

Previous evidence suggested an anti-oncogenic role for lysyl oxidase, mainly in ras-transformed cells. Here we prove that recombinant lysyl oxidase is actually able to antagonize p21-Ha-Ras-induced Xenopus laevis oocyte maturation. Lysyl oxidase was also effective on progesterone-dependent maturation, indicating a block lying downstream of Ras. Maturation induced by activated 'maturation promoting factor', normally triggered by progesterone, was also inhibited by lysyl oxidase. Finally, lysyl oxidase did not abolish p42Erk2 phosphorylation upon maturation triggering, suggesting a block downstream of Erk2. Further investigation showed that lysyl oxidase action depends on protein synthesis and is therefore probably mediated by a newly synthesized protein.

Aminopropionitrile↗
PubMed1997

Choline kinase inhibitors as a novel approach for antiproliferative drug design.

Recent progress in deciphering the molecular basis of carcinogenesis is of utmost importance to the development of new anticancer strategies. To this end, it is essential to understand the regulation of both normal cell proliferation and its alterations in cancer cells. We have previously demonstrated that in ras-transformed cells there is an increased level of phosphorylcholine (PCho) resulting from a constitutive activation on choiline kinase (ChoK). The importance of ChoK for the regulation of cell proliferation has also been proposed since an inhibitor for this enzyme, hemicholinium-3 (HC-3), drastically reduces entry into the S phase after stimulation with growth factors. Here we report the synthesis of several new compounds which are highly specific inhibitors for ChoK, with up to 1000-fold or 600-fold increased inhibitory activity, compared to HC-3 under ex vivo or in vitro conditions respectively. These novel compounds also drastically reduce entry into the S phase after stimulation with specific growth factors. A more profound inhibition of cell proliferation was observed in ras-, src- and mos-transformed cells in the presence of ChoK inhibitors, compared to their parental, untransformed NIH3T3 cells. By contrast, this effect was not observed in fos-transformed cells. While ras, src and mos transformation is associated with elevated levels of ChoK activity, fos-induced transformation does not affect ChoK activity. The inhibitory effect on proliferation of the new compounds correlates with their ability to inhibit the production of phosphorylcholine in whole cells, a proposed novel second messenger for cell proliferation. These results strongly support a critical role of choline kinase in the regulation of cell growth and makes this enzyme a novel target for the design of new antiproliferative and anticancer drugs.

3T3 Cells↗
PubMed1997

Signaling from G protein-coupled receptors to the c-jun promoter involves the MEF2 transcription factor. Evidence for a novel c-jun amino-terminal kinase-independent pathway.

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun promoter. In this study, we asked whether signals activating JNK regulate the c-jun promoter. Using NIH 3T3 cells expressing G protein-coupled m1 acetylcholine receptors as an experimental model, we have recently shown that the cholinergic agonist carbachol, but not platelet-derived growth factor, potently elevates JNK activity. Consistent with these findings, carbachol, but not platelet-derived growth factor, increased the activity of a c-jun promoter-driven reporter gene (for chloramphenicol acetyltransferase). However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but resulted in surprisingly limited induction of the c-jun promoter. This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore which of its regulatory elements participate in transcriptional control. We observed that deletion of the 3' AP-1 site diminished chloramphenicol acetyltransferase activity in response to carbachol, but only to a limited extent. In contrast, deletion of a MEF2 site dramatically reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the c-jun promoter in response to carbachol, and stimulation of m1 receptors, but not direct JNK activation, induced expression of a MEF2-responsive plasmid. Taken together, these data strongly suggest that MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a yet to be identified pathway, distinct from that of JNK.

3T3 Cells↗
PubMed1997

Regulation of proliferation and apoptosis by Ras and Rho GTPases through specific phospholipid-dependent signaling.

Small GTPases are molecular switches that control signaling pathways critical for diverse cellular functions. Recent evidence indicates that multiple effector molecules can be activated by small GTPases. As a result, complex biological processes such as cell proliferation and apoptosis are turned on. Thus, rather than a single linear pathway from the membrane to the nucleus, the integration of complementary signals is required for these events to occur. In fact, the coordinated activation of small GTPases may constitute some of the critical modulators of those signals triggering either proliferation or cell death. In addition to the activation of specific kinases cascades, phospholipid-derived messengers are candidates to compose some of the most critical elements associated to regulation of signaling cascades capable of discerning among life and death. Both proliferation and apoptosis needs competence and progression signals. Phospholipase D and sphingomyelinase may be important players in this decision-maker step.

Animals↗
PubMed1997

Activation of phospholipase D by growth factors and oncogenes in murine fibroblasts follow alternative but cross-talking pathways.

Phospholipase D (PLD) is activated by a variety of stimuli, including mitogenic stimulation by growth factors and oncogene transformation. Activation of PLD by growth factors requires protein kinase C (PKC) since depletion of the enzyme by down-regulation or direct inhibition by specific drugs completely abrogates this effect. Transformation by the ras and src oncogenes is also associated with an increase in basal PLD activity. However, this effect is not dependent on PKC, suggesting that growth factors and oncogenes may activate PLD by two independent mechanisms. Here we demonstrate that activation of PLD by phorbol esters is greatly enhanced in ras-transformed cells, suggesting synergistic activation of PLD by ras oncogenes and PKC. Also, ras-transformed cells showed a dramatic attenuation of the PLD activation induced by growth factors, although receptor function was still detectable. This attenuation paralleled the specific uncoupling of the phosphatidylinositol-specific phospholipase C (PI-PLC) pathway, indicating that activation of PLD by growth factors may be mediated by PI-PLC and PKC activation. Attenuation of PLD activation by platelet-derived growth factor was also observed in several oncogene-transformed cells, as well as the uncoupling of the PI-PLC pathway. Neither the co-operation with PKC activation nor the attenuation of the PLD response to growth factors in ras-transformed cells was a general consequence of cell transformation, since cells transformed by other oncogenes showed a normal response to either treatment. These results support the existence of at least two alternative signalling routes for the activation of PLD, one mediated by the PI-PLC/diacylglycerol/PKC pathway and a second one mediated by several oncogenes, independent of the PKC pathway, which synergizes with the PI-PLC/PKC-dependent pathway.

3T3 Cells↗
PubMed1997

Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins.

The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor kappaB (NF-kappaB) by a mechanism that involves phosphorylation of Ikappa Balpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-kappaB by TNFalpha depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-kappaB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-kappaB-dependent transcription.

3T3 Cells↗
PubMed1996

Activation of phospholipase D by ras proteins is independent of protein kinase C.

Growth factors activate phospholipases, causing the generation of diverse lipid metabolites with second messenger function. Among them, the phosphatidylcholine-preferring phospholipase D (PLD) has attracted great interest, since in addition to the transient activation by growth factors stimulation, it is constitutively activated in some of the src- and ras-transformed cells investigated. To establish further the functional relationship of ras oncogenes with PLD, we have investigated its mechanism of regulation. Growth factors such as PDGF or FGF activate the PC-PLD enzyme by a common, PKC-dependent mechanism. By contrast, ras oncogenes activate the PC-PLD enzyme by a PKC-independent mechanism. These results suggest that existence of at least two mechanisms for PLD activation, and ras oncogenes contribute to one of them.

3T3 Cells↗
PubMed1995

Induction of apoptosis by rho in NIH 3T3 cells requires two complementary signals. Ceramides function as a progression factor for apoptosis.

We have previously reported that rho genes, members of the ras superfamily, are tumorigenic when overexpressed in NIH 3T3 cells. As other known oncogenes, they also induce apoptosis after serum deprivation but not in the presence of growth factors. In the present study, we provide evidence that overexpression of the Aplysia Rho protein in NIH 3T3 cells induces the generation of phosphatidylcholine (PC)-derived second messengers as a result of activation of a PC-specific phospholipase D (PC-PLD) as previously reported for ras-transformed cells. In contrast, removal of serum in the Rho transfectants, but not in normal NIH 3T3 cells or cells transformed by the ras oncogene, induced the production of ceramides as a result of activation of an sphingomyelinase (SMase). Furthermore, the rho-expressing cells underwent apoptosis in the presence of serum when exogenous ceramides were added, and this process was accelerated if cells were treated with exogenous SMase. Thus, Rho proteins act as an initiation signal that is necessary but not sufficient for the induction of apoptosis in NIH 3T3 cells. We propose here that induction of apoptosis in NIH 3T3 cells requires two complementary signals: an initiation signal generated even in the presence of serum which 'primes' the cells, making them sensitive to a progression signal, triggered by serum removal, which we have identified as generation of ceramides.

3T3 Cells↗
PubMed1995

Evidence for different signalling pathways of PKC zeta and ras-p21 in Xenopus oocytes.

Considerable effort has been devoted to identifying critical steps in mitogenic signal transduction pathways. Recently, the atypical PKC zeta isoform has attracted great interest since it has been reported to induce GVBD in Xenopus oocytes and transformation of NIH3T3 fibroblasts, two processes closely linked with the regulation of cell division. Furthermore, PKC zeta has been proposed as an essential effector for ras-p21 function and therefore may be an essential component of the signalling pathway(s) activated by mitogens. In this study we have analysed the responses induced in Xenopus oocytes after microinjection of purified recombinant PKC zeta protein. Microinjection of PKC zeta induced the early activation of MPF which precedes GVBD and also induced the activation of MAP kinase and S6 kinase II. The activation of MPF, MAP kinase and S6 kinase II by PKC zeta was sensitive to cycloheximide, while induction of GVBD was independent of protein synthesis. These results indicate that PKC zeta induces the activation of at least two pathways, only one of them leading to the activation of MAP kinase. By contrast, neither the induction of GVBD nor the activation of MPF, MAPK and S6 kinase II induced by the ras-p21 protein were dependent on protein synthesis. Thus, the comparison of these responses suggests that PKC zeta most likely does not mediate the ras-induced signal transduction pathway in Xenopus laevis oocytes.

Animals↗
PubMed1995

Overexpression of PKC zeta in NIH3T3 cells does not induce cell transformation nor tumorigenicity and does not alter NF kappa B activity.

Signal transduction is the major mechanism by which cells communicate among themselves through extracellular stimuli. Among the different structural components involved in signal transduction, protein kinases are one of the key elements in the process. Protein kinase C is a multimember family of kinases which has been involved in the regulation of diverse cellular functions. Regulation of cell growth in fibroblasts has been reported to be one of such functions. In particular the PKC zeta isoenzyme has been postulated to be transforming to NIH3T3 cells (Berra et al., 1993) and to serve as an effector for Ras proteins through the regulation of the NF kappa B transcription factor (Dominguez et al., 1993) and direct interaction (Díaz-Meco et al., 1994). We have investigated the effects of overexpressing the mouse wild-type PKC zeta in NIH3T3 cells. When compared to the parental NIH3T3 cells, we have found (1) no significant effect on cell morphology; (2) no difference in growth properties in the absence of serum or in the presence of individual growth factors such as insulin, phorbol esters or PDGF; (3) no growth in soft agar nor tumorigenic activity in nude mice. In addition cells stably overexpressing the PKC zeta kinase did not interfere or amplify the induction of NF kappa B activity by tumor necrosis factor alfa (TNF-alpha) nor altered NF kappa B activity in transient expression of cells treated with TNF-alpha. Thus, mammalian PKC zeta is most likely not directly involved in the regulation of cell proliferation in fibroblasts nor affects directly or indirectly the activation of NF kappa B.

3T3 Cells↗
PubMed1995

Induction of apoptosis in NIH3T3 cells after serum deprivation by overexpression of rho-p21, a GTPase protein of the ras superfamily.

Oncogenes appear to influence apoptosis in two ways. Some activate cells from a growth-arrested state to one in which both apoptosis and entry to S-phase become possible, the choice between them being determined by a second signal, such as cytokine or growth factor. Cells in this state are often sensitive to apoptosis induced by a wide variety of agents, including several drugs used in cancer chemotherapy. Other oncogenes prevent activation of the apoptosis effector pathway, even in the presence of a death stimulus, the affected cells therefore being resistant to chemotherapeutic agents. In rodent fibroblasts, c-myc or the adenovirus oncogene E1A effect the first type of change, whereas bcl-2, v-abl, E1B or activated ras effect the second. Here we study in rodent fibroblast the effect of expression of rho genes, members of the ras superfamily which we have previously shown to be tumorigenic when highly expressed in this cell type. We show that expression of wild-type rho from Aplysia californica stimulates apoptosis in cultured cell lines and that the apoptotic index in tumors generated by these cell lines is similar to those induced by E1A-transformed cells. In contrast, mutated rho, activated by Val14 substitution in the GTP binding site, it less potent as a stimulator of apoptosis, generating a phenotype more similar to that obtained with activated ras. Thus, rho genes may play a critical role in the regulation of apoptosis.

3T3 Cells↗
PubMed1995

Modulation of cellular chemoresistance in keratinocytes by activation of different oncogenes.

Response to chemotherapeutic agents in malignant tumors depends on many factors, most of which are as yet unknown. We investigated the correlation between the activation of different oncogenes and protein-kinase-C (PKC) modulation, and the cytotoxicity of some of the most widely used anti-cancer drugs. We transformed the murine keratinocyte cell line PAM 212, with different oncogenes (v-H-ras, v-myc and adenovirus E1a) and a mutant p53 suppressor gene (mp53). The cytotoxic effect of cisplatin (CDDP), doxorubicin (DOX) and vincristine (VCR), together with the concomitant action of modulators of PKC, TPA and staurosporine were evaluated by the crystal-violet method, thymidine incorporation and flow cytometry. We report that (a) the oncogene v-H-ras induces resistance to CDDP (> 50%), DOX (> 25%) and VCR (> 20%); (b) the E1a oncogene induces only resistance to VCR (> 40%) and marked sensitivity to CDDP and DOX; (c) the mp53 oncogene induces more resistance to VCR and insignificant resistance to the other drugs; and (d) activation of PKC by TPA increases the resistance to VCR and DOX in cells transformed by the v-H-ras, while it significantly increases the lethality with CDDP of the E1a-transformed cells. Staurosporine increases the cytoxicity of all the drugs, especially in the E1a-transformed keratinocytes. In the flow-cytometry analysis, the percentage of BUdR incorporation was related to sensitivity to anti-cancer drugs.

Animals↗
PubMed1995

Generation of phosphorylcholine as an essential event in the activation of Raf-1 and MAP-kinases in growth factors-induced mitogenic stimulation.

Cell proliferation is regulated by an appropriate combination of intracellular signals involving activation of kinases and the generation of phospholipid metabolites. We report here that growth factors induce a biphasic generation of phosphorylcholine (PCho) in quiescent NIH 3T3 cells, resulting in an early and transient increase at 100 s and a larger and sustained increase after 3 h of stimulation. Generation of PCho at both early and late times of growth factors stimulation results from the consecutive activation of phospholipase D (PLD) and choline kinase (ChoK). Production of PCho by specific growth factors seems an essential requirement for the early signals associated to activation of Raf-1 and MAP kinases, since blockage of choline kinase completely inhibited activation of Raf-1 and MAP kinases by PDGF or FGF. Both the transient early increase and the late sustained increase in PCho are required for the induction of DNA-synthesis, besides completion of the activation of the serine/threonine kinases cascade. Thus, our results strongly suggest that generation of PCho by the PLD/choline kinase pathway is one of the critical steps in regulating cell growth in NIH 3T3 stimulated by growth factors.

3T3 Cells↗
PubMed1995

Activation of intracellular kinases in Xenopus oocytes by p21ras and phospholipases: a comparative study.

Signal transduction induced by generations of second messengers from membrane phospholipids is a major regulatory mechanism in the control of cell proliferation. Indeed, oncogenic p21ras alters the intracellular levels of phospholipid metabolites in both mammalian cells and Xenopus oocytes. However, it is still controversial whether this alteration it is biologically significant. We have analyzed the ras-induced signal transduction pathway in Xenopus oocytes and have correlated its mechanism of activation with that of the three most relevant phospholipases (PLs). After microinjection, ras-p21 induces a rapid PLD activation followed by a late PLA2 activation. By contrast, phosphatidylcholine-specific PLC was not activated under similar conditions. When each of these PLs was studied for its ability to activate intracellular signalling kinases, all of them were found to activate maturation-promoting factor efficiently. However, only PLD was able to activate MAP kinase and S6 kinase II, a similar pattern to that induced by p21ras proteins. Thus, the comparison of activated enzymes after microinjection of p21ras or PLs indicated that only PLD microinjection mimetized p21ras signalling. Finally, inhibition of the endogenous PLD activity by neomycin substantially reduced the biological activity of p21ras. All these results suggest that PLD activation may constitute a relevant step in ras-induced germinal vesicle breakdown in Xenopus oocytes.

Animals↗
PubMed1994

ras-p21 activates phospholipase D and A2, but not phospholipase C or PKC, in Xenopus laevis oocytes.

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.

Amino Acid Sequence↗