Human Molecular Genetics, 2001, Vol. 10, No. 17 1741-1752
© 2001 Oxford University Press
Fumarylacetoacetate, the metabolite accumulating in hereditary tyrosinemia, activates the ERK pathway and induces mitotic abnormalities and genomic instability
Laboratory of Cell and Developmental Genetics, Department of Medicine, Pav. C.-E. Marchand, Université Laval and CHUL Research Center, Sainte-Foy, Quebec, G1K 7P4, Canada
Received February 8, 2001; Revised and Accepted June 19, 2001.
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ABSTRACT |
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Patients suffering from the metabolic disease hereditary tyrosinemia type I (HT1), caused by fumarylacetoacetate hydrolase deficiency, have a high risk of developing liver cancer. We report that a sub-apoptogenic dose of fumarylacetoacetate (FAA), the mutagenic metabolite accumulating in HT1, induces spindle disturbances and segregational defects in both rodent and human cells. Mitotic abnormalities, such as distorted spindles, lagging chromosomes, anaphase/telophase chromatin bridges, aberrant karyokinesis/cytokinesis and multinucleation were observed. Some mitotic asters displayed a large pericentriolar material cloud and/or altered distribution of the spindle pole-associated protein NuMA. FAA-treated cells developed micronuclei which were predominantly CREST-positive, suggesting chromosomal instability. The Golgi complex was rapidly disrupted by FAA, without evident microtubules/tubulin alterations, and a sustained activation of the extracellular signal-regulated protein kinase (ERK) was also observed. Primary skin fibroblasts derived from HT1 patients, not exogenously treated with FAA, showed similar mitotic-derived alterations and ERK activation. Biochemical data suggest that FAA causes ERK activation through a thiol-regulated and tyrosine kinase-dependent, but growth factor receptor- and protein kinase C-independent pathway. Pre-treatment with the MEK inhibitor PD98059 and the Ras farnesylation inhibitor B581 decreased the formation of CREST-positive micronuclei by
75%, confirming the partial contribution of the Ras/ERK effector pathway to the induction of chromosomal instability by FAA. Replenishment of intracellular glutathione (GSH) with GSH monoethylester abolished ERK activation and reduced the chromosomal instability induced by FAA by 80%. Together these results confirm and extend the previously reported genetic instability occurring in cells from HT1 patients and allow us to speculate that this tumorigenic-related phenomenon may rely on the biochemical/cellular effects of FAA as a thiol-reacting and organelle/mitotic spindle-disturbing agent. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Among diseases affecting the tyrosine degradation pathway, hereditary tyrosinemia type 1 (HT1; McKusick No. 276700) presents the most severe symptoms mainly affecting the liver. These include progressive hepatic dysfunction at early infancy and cirrhosis/hepatocellular carcinoma at mid-childhood (for a review see 1). Liver cancer has been reported in 37% of patients older than 2 years and held responsible for 16% of HT1 mortality (2). This autosomally recessive inherited disease is caused by the deficiency of fumarylacetoacetate hydrolase (FAH; EC 3.7.1.2) (3,4), the enzyme responsible for the conversion of fumarylacetoacetate (FAA) in fumarate and acetoacetate. The functionality of this catalytic step seems important for the normal physiological state of the hepatocyte since we and other investigators have demonstrated that FAA, an
,ß-unsaturated carbonyl compound, reacts with glutathione (GSH) and protein thiol groups (5–7) and displays mutagenic, cytostatic and apoptogenic activities (8–10). Cell cycle- and mitochondrial dysfunction-related events as well as caspase activation are among the molecular events underlying FAA-induced apoptosis, which is modulated by intracellular GSH levels and caspase inhibitors (10–12). Thus, the ability of FAA to induce apoptosis in vitro (10) and also likely in vivo (11,12), at high doses or under conditions of intracellular GSH depletion, seems to be at the basis of the acute liver failure observed in HT1. However, the molecular basis for the high incidence of liver cancer in HT1 remains less understood. Two studies using biological material derived from HT1 patients have shown the occurrence of cellular alterations reflecting genetic instability, such as chromosome breakage in skin fibroblasts (13) and aneuploidy in hepatocytes (14). Using cultured rodent V79 cells, we have shown that FAA is mutagenic and that GSH depletion can potentiate the mutagenicity of FAA (8,9). This could be taken as evidence for direct genetic damage caused by FAA, thus suggesting that FAA may initiate the carcinogenic process in HT1. However, the possibility that FAA may contribute to other steps of this process, such as in tumor promotion, could not be excluded. In this regard, Onfelt (15) has demonstrated that GSH-reacting agents such as diethylmaleate (DEM), which, like FAA, depletes GSH by adduction, generally act as spindle-disturbing agents causing c-mitosis. Indeed, induction of c-mitosis and modifications of the fidelity of cellular division by agents that affect the mitotic spindle have been shown to cause aneuploidy (16). Moreover, it has been demonstrated that a significant proportion of chemical aneugens are potential complete carcinogens, thus meaning that aneuploidy may contribute to tumor promotion after a mutagenic, initiating insult (16). However, the GSH/thiol-targeting activity of FAA should be expected to directly or indirectly impair a vast array of important cellular processes, given the well known involvement of thiol groups in the regulation of numerous proteins/enzymes, as well as in redox regulation, to which many signaling molecules are subjected (for a review see ref. 17). For example, all known protein tyrosine phosphatases are -SH enzymes and it has been demonstrated that those specifically involved in the dephosphorylation of tyrosine kinases mediating receptor-stimulated signaling pathways are commonly inactivated by thiol-alkylating or thiol-oxidizing agents (18). Indeed, inhibition of protein phosphatases causing the activation of mitogenic signaling cascades, such as those involving Jun N-terminal kinases (JNKs), seems to underlie the tumor-promoting activity of the thiol-reacting agent arsenite (19).
In mammalian cells, the extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, is crucial for regulating normal cell growth and differentiation (20,21) and is preferentially activated by mitogens and growth factors (22). ERK activation generally proceeds through the sequential activation of the module Raf
MEKERK (for a review see ref. 23) after the initial activation of receptor or non-receptor (cytosolic) tyrosine kinases and Ras activation (ref. 24 and references therein). However, activation of Raf/ERK through Ras-independent pathways can be induced in some cell types by tumor promoters, such as phorbol ester or okadaic acid, acting respectively through direct activation of Raf by protein kinase C (PKC) or inhibition of MEK/ERK dephosphorylation by inhibition of serine/threonine phosphatases (25,26). In cultured cells, dysregulation of the ERK pathway due to alterations in any of the several mediators involved in the cascade, such as Ras, Raf or MEK, leads to cellular transformation (27,28). Indeed, elevated ERK activity is found in many human tumors, including hepatocellular carcinoma (29). In rodent cells, increased constitutive ERK activity resulting from Ras or Mos oncogene expression leads to genomic instability underlying on altered microtubule dynamics and/or spindle disturbances and characterized by mitotic aberrations and the generation of whole chromosome-containing micronuclei (30,31).
In the present study, we examined the possibility that FAA, acting as a GSH/thiol-reacting agent, could induce spindle disturbances that might result in genomic instability in cultured mammalian cells. We found that a sub-apoptogenic, acute dose of FAA induced mitotic abnormalities and caused chromosomal instability, with predominance of aneugenic over clastogenic events, as assessed by differential CREST-labeling of induced micronuclei. At the biochemical level, FAA caused activation of the Ras/ERK pathway through mechanism(s) involving GSH/thiol-dependent events. We demonstrated that activation of this mitogenic pathway contributed by up to 75% to chromosomal instability induced by FAA, an effect that showed a high (80%) dependency on intracellular GSH levels. As mitotic aberrations, chromosomal instability and sustained ERK activation were also observed in skin fibroblasts derived from HT1 patients (not exogenously treated with FAA), a putative role for the in vitro observed cellular and biochemical effects of FAA is discussed in relation to genomic instability and the carcinogenic process in HT1.
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RESULTS |
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FAA induces mitotic abnormalities and chromosomal instability
We have previously shown that in mammalian cells treated with FAA in Hank’s balanced salt solution (HBSS) for 2 h and then left to recover, FAA induces G2/M arrest (24 h post-treatment) and massive apoptosis (48 h post-treatment). The magnitude of the cytostatic and apoptotic effects of FAA were dose-dependent and related to the levels of intracellular GSH (10). Here we used the maximal dose of FAA assayed in our previous study, i.e. 100 µM, and the same time exposure (2 h) but FAA was added to the complete culture medium. This pulse treatment protocol induced neither an irreversible cell cycle block nor apoptosis in exponentially growing V79 or Hela cells, causing only a slight accumulation of cells in G2/M (12.5% versus 9% in control V79 cells and 13% versus 10% in control Hela cells; see Fig. 1A for example of DNA histograms in Hela cells), which was evident immediately post-treatment, but not later (24 h) during recovery. The resulting slowed-down progression through G2/M was easily observed in synchronized cells treated with FAA (see Fig. 1B for example of DNA histograms in V79 cells). This allowed us to examine cells during and after their mitotic exit in search for FAA-induced morphological alterations.
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Analysis by Giemsa staining of exponentially growing V79 or Hela cells treated with FAA showed many mitotic abnormalities such as spindle disturbances, lagging chromosomes in metaphase and anaphase cells, aberrant karyokinesis/cytokinesis and segregational defects, as well as micronuclei and multinucleated cell formation (Fig. 2A–I). Mitotic abnormalities were predominantly observed immediately after FAA treatment, with as many as 56% of metaphase and 45% of anaphase/telophase V79 cells showing abnormal characteristics (Table 1). Micronucleation (21%) and multinucleation (5%) predominantly occurred later during recovery (24–48 h post-treatment). Similar results were found in Hela cells (data not shown). Interestingly, similar mitotic-related alterations were observed in primary skin fibroblasts derived from HT1 patients (not exogenously treated with FAA) (Fig. 2J and K). These results suggest that FAA acts as a spindle-disturbing agent and also alters the coordination of karyokinesis and cytokinesis.
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To better understand the basis for FAA-induced mitotic abnormalities, synchronized V79 or Hela cells in late S-early G2 phase were pulse treated with FAA and examined during or after exit from mitosis by immunofluorescence microscopy. Early after treatment, when untreated cells had already progressed into the next G1 phase, FAA-treated cells were still progressing through mitosis (Fig. 1B), but almost no cells with normal mitotic phase morphology were observed. As shown in Figure 3, the majority of cells were in a prometaphase-like state or metaphase with fragmented (Fig. 3A and A'), distorted (Fig. 3B and B'), monopolar or multipolar spindles (Fig. 3C and C'), as evidenced with an anti-ß-tubulin antibody, and anaphase/telophase chromatin bridges were frequently observed in Hoescht-counterstained cells. Abnormal segregation of chromatin also generated cytoplast daughter cells (Fig. 3C and C'). Asynchrony of mitotic events was also observed in some cells in telophase where one of two daughter cells displayed a metaphasic spindle in contrast to condensed chromatin in the other one (Fig. 3D and D'). Multinucleation (as evidenced by NuMA and Hoescht labeling; Fig. 3G' and G'') was frequent in late post-mitotic cells (24 h post-FAA treatment) which displayed an almost normal microtubule arrangement (Fig. 3G), but these multinucleated cells were non-viable at 48–72 h post-treatment (data not shown). Although centrosome components, i.e. centrioles, as evidenced using anti-
-tubulin antibody, and pericentriolar material (PCM) remained generally associated, some mitotic asters displayed multiple centrosomes and/or large PCM cloud (Fig. 3E, E' and F') and/or altered distribution of the spindle pole-associated protein NuMA (Fig. 3G'). This protein, with nuclear localization in interphasic cells, is normally present at the metaphasic spindle poles with a characteristic ‘V’ shaped pattern of distribution (Fig. 3G', inset), reflecting localization at centrosome and both the minus and plus ends of spindle microtubules. In the mitotic asters of FAA-treated cells, NuMA showed rather a diffuse distribution (Fig. 3G').
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Since our morphological results using Giemsa staining, or immunofluorescence analysis of cells counterstained with Hoescht to visualize DNA, showed that, beside other mitotic abnormalities, micronuclei were formed in FAA-treated cells, we further examined the generation of micronuclei in these cells. Micronucleus formation reflects damage induced by agents that disrupt the mitotic spindle or that induce double-strand DNA breaks, consequently leading to micronuclei which contain whole chromosomes not incorporated into the daughter nuclei or micronuclei containing acentric chromosome fragments, respectively. In order to distinguish between these two types of micronuclei, an immunofluorescence analysis of FAA-treated cells was performed with the anti-kinetochore antibody CREST, which recognizes centromeric proteins. An example of CREST-positive and CREST-negative micronuclei staining with this antibody is presented in Figure 3H and H'. Staining of cells with this antibody revealed that CREST-positive micronuclei predominated (
70%) in V79 or Hela cells treated with FAA. Similar results were obtained after CREST staining of human normal fibroblasts treated with FAA or HT1 fibroblasts not exogenously treated with FAA. The predominance of CREST-positive micronuclei after FAA treatment of cells was thus considered as evidence for mitotic instability due to spindle disturbances induced by FAA, resulting in loss of whole missegregated chromosomes, characteristic of the action of aneugenic agents. The generation of CREST-negative micronuclei reflected the loss of acentric chromosome fragments, suggesting that FAA was also acting as a clastogenic agent. Thus, FAA basically seems to cause mitotic aberrations leading to chromosomal instability by altering the spindle morphology through its direct or indirect action on spindle-associated proteins and also by inducing asynchronous karyokinetic/cytokinetic events.
FAA does not directly attack cellular microtubules, but causes a rapid disruption of the Golgi apparatus
Given the presence of distorted spindles in FAA-treated cells and since agents that alter the mitotic spindle could act by directly targeting the main component of cellular microtubules, tubulin, we determined the relative levels of soluble and polymerized tubulin in V79 or Hela cells treated with FAA. As shown in Figure 4A, western blot analysis using an antibody against ß-tubulin showed no changes in the levels of free tubulin or tubulin present in microtubule fractions of V79 cells treated with FAA (100 µM for 2 h), compared with control cells. In contrast, extensive tubulin solubilization was observed after treatment of cells with the microtubule-depolymerizing agent nocodazole (Fig. 4A). Similar results were observed in Hela cells or after treatment with FAA during 24 h (data not shown). Thus, FAA did not seem to directly affect the polymerization or depolymerization status of cellular microtubules. However, FAA effects on the mitotic spindle might be mediated by attack of other cellular structures that are in direct relation with microtubule organization. One such organelle is the Golgi apparatus, which plays a key role in microtubule/mitotic aster organization through interaction with the endoplasmic reticulum (ER) and motor proteins (32). Thus, FAA-treated cells were analyzed by immunofluorescence microscopy using a human autoimmune serum that recognized the Golgi-associated autoantigen Golgin-97 (33). As shown in Figure 4B, FAA treatment caused a rapid (within 2 h) disruption of the Golgi complex, an effect that was clearly evident in interphase cells (Fig. 4Bb) where the Golgi complex appeared randomly distributed within the cell, in contrast to control cells where it appeared more or less unified juxtanuclearly (Fig. 4Ba). No evident microtubule alterations were observed in FAA-treated cells (Fig. 4Bb') compared with control cells (Fig. 4Ba'). For comparison, an example of cells treated with nocodazole, which also extensively disrupts the Golgi apparatus, is also shown (Fig. 4Bc and c'). A lower degree of Golgi disruption was observed at 24 h post-FAA treatment (data not shown). Interestingly, similar Golgi alterations were observed in HT1 fibroblasts without any exogenous FAA treatment (Fig. 4Bd). Thus, it seems possible that massive Golgi disruption caused by FAA could affect cell division indirectly by impairing the role of Golgi elements on the early organization and maintenance of a specific microtubule arrays, such as the mitotic spindle. Although no evident alterations in the reticular staining pattern of the ER were observed in FAA-treated Hela cells by immunofluorescence analysis using an antibody against VASAP-60 (34), some alterations (loss of labeling) were observed at the level of vesicular-like structures localized in the perinuclear region and throughout the cytoplasm (data not shown).
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FAA causes activation of the Ras/ERK pathway through mechanism(s) highly dependent on intracellular GSH
Since cellular alterations such as mitotic abnormalities, chromosomal instability and Golgi disruption have been shown to occur in cell systems with increased ERK activity (30,31,35,36), we evaluated whether the ERK pathway was activated by FAA. For this purpose, V79 and Hela cells were treated with FAA and lysates prepared immediately after treatment or, after changing to fresh medium, cells were left to recover for different time periods. ERK activation was then assessed by western blotting using an antibody against active ERK, i.e. against phosphorylated ERK1/2. As shown in Figure 5A, FAA activated ERK in both cell lines, but this activation was stronger and more rapid in V79 cells than in Hela cells. ERK activation was characterized by a predominance of ERK2 (p42) phosphorylation. It was seen immediately after the end of FAA treatment, maintained until 24 h post-treatment and decreased thereafter. No changes in JNK/stress-activated protein kinase (SAPK) or p38 MAPK basal phosphorylation levels were induced by FAA (data not shown). Interestingly, the greater response of rodent cells for ERK activation compared with human cells was also observed in liver cancer-derived cell lines treated with FAA, such as in Hepa1c1c7 (murine hepatoma) cells versus HepG2 (human hepatocarcinoma), and ERK activation in both cell types was maintained until 48 h post-FAA treatment (data not shown). It is noteworthy that high levels of phosphorylated ERK were also observed in HT1 fibroblasts (not exogenously treated with FAA) (Fig. 5B). Among other tyrosine metabolites, neither maleylacetoacetate (100 µM x 2 h) nor succinylacetone (1 mM x 2 h) induced ERK activation (data not shown).
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To determine which upstream mediators were involved in ERK activation by FAA, we first examined the effect of the MEK-specific inhibitor PD98059 on FAA-induced ERK phosphorylation in V79 cells. As shown in Figure 6, pre-treatment of cells with PD98059 greatly reduced FAA-induced ERK phosphorylation (by
65%). In cells treated with PD98059 alone, endogenous active ERK was completely abolished at the end of treatment, but returned to basal levels 3 h after treatment (not shown). The involvement of Ras as activator of the Raf/MEK/ERK module was then assessed by pre-treating cells with B581, an inhibitor of Ras farnesylation (an obligatory step in Ras processing). As shown in Figure 6, the level of ERK phosphorylation induced by FAA is reduced almost to control values after pre-treatment of cells with this inhibitor. However, since B581 was able to completely block endogenous ERK activation in cells not treated with FAA (not shown), its inhibitory effect on FAA-induced ERK phosphorylation needs to be considered only as partial. These data and those obtained with PD98059 suggest that, besides Ras activation, a strong signal or effect induced by FAA contributes to maintenance of ERK activation, which takes place somewhere in the Raf/MEK/ERK module.
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Since Raf activation can be Ras-independent and results from direct activation by mediators such as PKC or can be regulated by cAMP through activation of protein kinase A (PKA), a negative regulator of Raf activity, we determined the involvement of these mediators in FAA-induced ERK activation by pre-treating V79 cells with the PKC inhibitor staurosporine or the adenylate cyclase activator forskolin. No significant effect on ERK activation by FAA was observed in staurosporine-pre-treated cells, but a small, although significant decrease in ERK phosphorylation (by
20%) was observed in forskolin-pre-treated cells (Fig. 6). These results thus confirm the involvement of Raf in the FAA-induced ERK activation pathway and the possibility of modulation of Raf activity via cAMP/PKA, but exclude PKC as direct activator of the Raf/MEK/ERK module.
Since Ras activation generally occurs by tyrosine phosphorylation of Shc-, Src- or Src-related proteins through tyrosine kinases associated with both receptor and non-receptor proteins, we determined whether tyrosine kinases are involved in FAA-induced ERK activation. Pre-treatment of V79 cells with genistein, a broad tyrosine kinase inhibitor, reduced FAA-induced ERK phosphorylation by 30% (Fig. 6). Thus, tyrosine kinases are mediators localized upstream of Ras/Raf in the pathway leading to ERK activation by FAA.
To directly examine the role of growth factor receptors in general, or that of epidermal growth factor receptor (EGFR) in particular, in mediating FAA-induced ERK activation, we co-treated V79 cells with FAA and the inhibitory agent suramin, a general growth factor receptor antagonist, or tyrphostin AG1478, a selective EGFR kinase inhibitor. As shown in Figure 6, neither suramin nor tyrphostin AG1478 significantly changed the levels of ERK phosphorylation induced by FAA. Thus, these data indicate that in V79 cells, growth factor receptors (and their intrinsic associated tyrosine kinases) were not directly involved in triggering the signaling cascade leading to ERK activation after FAA treatment.
Since FAA is a thiol-reacting agent and given the known thiol- and redox-mediated regulation of several tyrosine kinases and phosphatases involved in intracellular signaling pathways, we decided to investigate whether ERK activation by FAA could be affected by modulating intracellular GSH contents. For this purpose, we first analyzed whether a known GSH-depleting agent such as DEM, which conjugates GSH as does FAA (9), could activate ERK in V79 cells, as it has been previously reported to occur in DEM-treated Hela cells (37). As shown in Figure 6, V79 cells treated with DEM (0.5 mM x 2 h) showed increased levels of phosphorylated ERK, as observed after FAA treatment. DEM-induced ERK activation was also observed in HepG2 cells (not shown). Then we pre-treated V79 cells with glutathione-monoethylester (GSH-MEE) or N-acetylcysteine (NAC) in order to increase intracellular GSH levels prior to FAA treatment. As shown in Figure 7, FAA-induced ERK activation was almost completely inhibited in cells pre-treated with GSH-replenishing agents. Thus, these results suggest that thiol groups are critical components of effector proteins involved in the cascade leading to ERK activation by FAA and that, consequently, this signaling cascade can be modulated by intracellular GSH contents.
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The chromosomal instability induced by FAA is partially mediated by Ras/ERK activation and depends on intracellular GSH
Finally, since activation of Ras and/or ERK has been demonstrated to be sufficient to induce mitotic abnormalities and chromosomal instability (30,38), we analyzed the effect of several Ras/ERK modulators on chromosomal instability induced by FAA. For this purpose, V79 cells were pre-treated with PD98059, B581, wortmannin or GSH-MEE. As shown in Figure 7, the frequency of CREST-positive micronuclei induced by FAA was significantly decreased after pre-treatment with PD98059 (frequency decreased by 76%), B581 (decreased by 71%), wortmannin (decreased by 65%) or GSH-MEE (decreased by 80%). All inhibitor pre-treatments were without effect on the frequency of CREST-negative micronuclei induced by FAA and did not affect cell growth after 48 h post-treatment (data not shown). Thus, these results suggest that chromosomal instability induced by FAA partially results from the activation of the Ras/ERK effector pathway, but the Ras/PI3K as well as other unknown pathways also seem to contribute to this effect. Moreover, regardless of the specific mechanisms underlying the induction of chromosomal instability by FAA, this effect seems to be highly dependent on intracellular GSH levels or the reduced status of mediator molecules, since it was greatly reduced by pre-treatment of cells with the GSH-replenishing agent.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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By showing that FAA causes mitotic abnormalities in mammalian cells, our present results are in agreement with those of Onfelt (15,39) who reported that GSH-reacting agents generally act as spindle-disturbing agents causing partial or full c-mitosis. In addition, we demonstrate that FAA causes chromosomal instability, predominantly resulting from aberrant mitotic segregation and whole chromosome loss, as assessed by CREST-positive micronuclei formation. Thus, we provide evidence supporting the hypothesis that the cancer process in the liver of HT1 patients may lie on the activity of FAA as, on one side, a mutagen participating in the initiation step, and on the other side, an aneugen promoting tumor development through mechanism(s) partially involving the sustained activation of the ERK signaling pathway.
Although the cellular effects induced by FAA are essentially similar to those induced by oncogene (Ras or Mos)-mediated ERK activation (30,31,35), based upon our results with wortmannin, we cannot rule out the possibility that FAA-induced chromosomal instability may also result from activation of the Ras/PI3K effector pathway (40). Since increased Ras/ERK activity enhances genomic instability due to p53 deficiency (35,41), continuous activation of the Ras/ERK pathway might contribute to enhanced genetic instability in HT1 cells lacking normal p53 function. Indeed, decreased p53 levels have been observed in HT1 fibroblasts (R.Jorquera and R.M.Tanguay, unpublished data). The predominance of CREST-positive micronuclei in FAA-treated cultured cells as well as in HT1 fibroblasts (not exogenously treated with FAA) could be taken as direct evidence for chromosomal instability due to spindle disturbances induced by FAA, which results in loss of whole missegregated chromosomes and eventually in aneuploidy (14). Since we also observed a minor clastogenic effect of FAA, i.e. generation of micronuclei with acentric chromosome fragments, this could explain the previous finding of chromosome breakage in HT1 cells (13).
The finding that both ERK activation and chromosomal instability induced by FAA was markedly abolished when cells were pre-treated with NAC or GSH-MEE suggests that the GSH/thiol-reacting activity of FAA is involved in causing ERK activation and chromosomal instability, by depleting GSH or altering the intracellular redox state and critical SH-groups in regulatory proteins. Similar activation of ERK by GSH depletion has been shown in human cells after treatment with DEM (37), an agent that depletes intracellular GSH similarly to FAA, i.e. by adducting GSH (9). It has been demonstrated that intracellular thiol/GSH depletion correlates with apoptosis when p38 MAPK, but not ERK, is selectively activated in DEM-treated human fibroblasts (42). In the present study, the absence of FAA-induced apoptosis when cells were treated with FAA added to the complete culture medium certainly reflects the extracellular adduction of FAA with SH-containing molecules, thus lowering the effective FAA concentration that targets the cell. In fact, we have observed activation of p38 MAPK only when cells were treated with FAA in HBSS (R.Jorquera and R.M.Tanguay, unpublished data), a treatment that causes a severe GSH depletion (to 5% of control levels) and leads to cell cycle arrest-related apoptosis (10).
The partial decrease in ERK phosphorylation observed after cell pre-treatment with the MEK inhibitor PD98059 or the Ras-processing inhibitor B581 suggests either that these agents failed to completely prevent FAA-induced MEK/ERK activation, as occurs in the case of strong ERK activators like the nerve growth factor or epidermal growth factor (EGF) (43), or that an imbalance between ERK phosphorylation and ERK dephosphorylation is occurring. Activation of tyrosine kinases, excluding the one associated with EGFR, is certainly involved in the pathway leading to ERK activation by FAA, since pre-treatment of cells with genistein, a broad tyrosine kinase inhibitor, decreased ERK phosphorylation by 30%, while pre-treatment with tyrphostin AG1478 was without effect. However, the partial inhibition in ERK phosphorylation achieved by genistein seems to indicate that activation of tyrosine kinases is not the main mechanism leading to FAA-induced ERK activation. Although other studies have shown similar extents of reduction by genistein of ERK activation upon stimulation of G-protein-coupled receptors (GPCRs) (44,45), on the basis of our results showing resistance of FAA-induced ERK phosphorylation to suramin, it seems unlikely that GPCRs or other growth factor receptors are involved in the induction of ERK by FAA in V79 cells, since these receptors are generally suramin-inhibitable. Thus, additional event(s) leading to ERK activation by FAA, need to be considered, such as the direct activation of Raf by FAA, as it has been shown to occur after treatment of human and murine hepatoma cells with the thiol-reacting agent sulphorane (46), or the inhibition of protein phosphatases involved in the Ras/ERK pathway.
The direct or indirect inhibition of protein phosphatases by FAA could result in sustained ERK activity, as it has been shown to occur in arsenite-induced JNK activation (19). Arsenite, a thiol-reacting agent and also a tumor promoter, has been shown to stimulate JNK activity by inhibiting a constitutive dual-specificity JNK phosphatase, which appears to be responsible for maintaining low basal JNK activity in non-stimulated cells (19). Arsenite has also been shown to induce Ras-dependent ERK activation by a mechanism involving phosphorylation of EGFR and activation of the adaptor protein Shc (47). In this case, inhibition of tyrosine phosphatases involved in the inactivation of EGFR tyrosine kinase has been suggested as one possible mechanism leading to EGFR-mediated arsenite-induced ERK activation. In fact, the concerted activities of tyrosine kinases and phosphatases involved in the ERK pathway, like in other signaling pathways, have been shown to be sensitive to thiol- and redox-regulation (17,18). An example of this is the common inactivation of tyrosine phosphatases which dephosphorylate receptor tyrosine kinases by thiol-alkylating or thiol-oxidizing agents, as well as by radiation, an effect that can be reversed by NAC (18). FAA, as a thiol-reacting agent, may thus inhibit one or more phosphatases involved in the maintenance of low basal ERK activity in quiescent cells or in the inactivation of tyrosine kinases mediating FAA-induced ERK activation. Indeed, the high levels of ERK phosphorylation, i.e. sustained ERK activation, observed in fibroblasts derived from HT1 patients indirectly supports the occurrence of such events. In any case, the reduction in FAA-induced ERK phosphorylation observed after pre-treatment of V79 cells with NAC or GSH-MEE could be explained by the inhibitory effect of NAC on the activation of Src-related tyrosine kinases (48) or by the adduction of FAA by GSH, thus abolishing at a general level the effects of FAA that cause activation of the ERK pathway.
More indirect evidence that the balance between ERK phosphorylation and dephosphorylation activities may be affected by FAA is given by the similarities existing between the FAA-induced spindle disturbances/mitotic aberrations and those induced by agents impairing reversible phosphorylation reactions involved in mitosis regulation. In fact it has been shown that in V79 cells, tyrphostins or carbaryl, which inhibit tyrosine kinase activities and concomitantly induce serine/threonine phosphatase activities, also cause c-mitosis, displaced chromosomes and uncoordinated karyokinesis and cytokinesis (49). The imbalance between phosphorylation/dephosphorylation reactions induced by FAA may also affect microtubule dynamics, mitotic spindle morphogenesis and chromosome movements through functional impairment of microtubule-associated accessory proteins (50) and/or motor proteins (51), also leading to distorted spindles and missegregated chromosomes (52,53). In agreement with this, we found that NuMA, a spindle-associated protein that normally interacts with motor proteins to ensure proper spindle formation and functioning (54), showed altered distribution in FAA-treated cells.
Chemicals that cause sustained activation of constitutive mitogenic signaling cascades by, for example, inhibiting the activity of protein phosphatases, such as the serine/threonine protein phosphatase inhibitor okadaic acid, the thiol-reacting agent arsenite or the ER Ca2+-depleting agent thapsigargin, are indeed well known tumor promoters (19,55). The tumor promotion activity of these compounds seems to lie in part on their ability to activate MAPKs and consequently to activate nuclear transcription factors governing the expression of several genes involved in cell growth, differentiation and transformation, such as immediate-growth-response genes and AP-1-responsive genes (56,57). Elevated cytosolic Ca2+, through activation of calmodulin-dependent protein kinases and calcineurin, can also activate MAPKs and regulate the activity of such genes (58–60). Interestingly, we effectively observed a rise in cytosolic free Ca2+ after incubation of V79 or Hela cells with FAA during 2 h (data not shown) and the potential relationship of this effect with FAA-induced ERK activation is currently studied in our laboratory. The altered gene expression observed in human HT1 (61) as well as in murine HT1 models (62,63) may be thus directly or indirectly related to the increase in ERK activity caused by FAA. For example, in hepatocytes from murine HT1 models, increased expression of genes coding for phase II detoxifying enzymes, such as NAD(P)H:menadione oxidoreductase (NMO-1) or glutathione S-transferases, has been reported (62,64). The promoters of these genes have in common the presence of the anti-oxidant responsive element (ARE) which is similar to the AP-1-binding site sequence, thus making possible their activation by the AP-1 complex. Recently, Yu et al. (46) demonstrated that ERK activation mediates induction of ARE-dependent phase II detoxifying enzymes, such as NMO-1. Interestingly, we have also observed NMO-1 induction in FAA-treated V79 cells (R.Jorquera and R.M.Tanguay, unpublished data).
Another intriguing observation in the present study is the extensive Golgi disruption observed after FAA treatment of cultured cells as well as in HT1 fibroblasts not exogenously treated with FAA. In the c14CoS/c14CoS mouse model for HT1, both the ER and Golgi apparatus from parenchymal liver cells and proximal tubule kidney cells (the main tissues affected in human HT1) display profound ultrastructural alterations, which are corrected by expression of transgenic FAH (64). ER alterations in hepatocytes have also been reported in another murine model of HT1, the FAH knock-out mouse (62), and from our present data, we cannot exclude the possibility that FAA may cause subtle changes in the ER structure, such as those evidenced by electron microscopy in the HT1 mouse models. In mammalian cells, the Golgi and the ER serve as main stores for Ca2+, both organelles sharing many Ca2+ homeostatic properties, with SERCA pumps for active Ca2+ uptake and IP3 receptors for passive Ca2+ efflux (65,66). Thus, the structural changes of the Golgi complex induced by FAA may be potentially associated with functional alterations of this organelle, causing, for example, the release of stored Ca2+, which may contribute to increased intracellular cytosolic Ca2+ levels. This possibility is the subject of current investigations in our laboratory since both depletion of internal Ca2+ stores as well as increased cytosolic Ca2+ have been shown to cause activation of phospholipase A2 (67,68), which mediates Golgi disruption associated with cell transformation caused by oncogenic Ras (36). Moreover, hepatocytes from the c14CoS/c14CoS mouse HT1 model also show increased phospholipase A2 activity and arachidonic acid generation, suggesting an inflammatory response through cyclooxygenase-2 activation (46). Both phospholipase A2 and cyclooxygenase-2 are cytosolic substrates for active ERK and cyclooxygenase-2 is known to be induced by tumor promoters or oncogenes (69,70). Thus, events such as Golgi disruption, Ca2+ mobilization or inflammation could also be components of a broad range of cellular and biochemical responses occurring in cells exposed to FAA that ultimately may contribute to tumor initiation in HT1.
In conclusion, based upon our overall present and previous findings, we speculate that the carcinogenic process in the livers of HT1 patients may be initiated by the mutagenic activity of FAA and then tumor promotion could be partially favored by sustained FAA-induced ERK activation causing chromosomal instability, aneuploidy and transformation. We believe that the demonstrated in vitro ability of GSH-replenishing agents, such as the cell-permeant GSH-MEE, to attenuate main events potentially causally related to HT1-associated pathologies, such as apoptosis, mutagenesis and ERK activation (mitogenesis), merits further exploitation clinically for the benefit of HT1 patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Materials
FAA was synthesized as described previously (71) and kept frozen at –80°C until used. FAA was dissolved in HBSS (Gibco BRL Products, Burlington, Canada) supplemented with NaHCO3 (0.35 g/l), pH 7.3, and filter sterilized before treatment of cells. Its concentration was determined by spectral analysis (71). PD98059, suramin, tyrphostin AG1478, genistein, wortmannin, staurosporine, GSH-MEE, NAC and mimosine were purchased from Sigma Chemical (St Louis, MO). Forskolin was from ICN (Costa Mesa, CA). B581 was from Biomol (Plymouth Meeting, PA). Phosphospecific antibody for ERK1/2 was from New England Biolabs (Mississauga, Canada). Human autoimmune serum against NuMA, PCM and centromere/kinetochore (CREST) were gifts from Dr J.B.Rattner (University of Calgary, Calgary, Canada). Rabbit polyclonal anti-Golgin-97 was kindly provided by Dr M.Fritzler (University of Calgary, Calgary, Canada). Monoclonal antibody against ß-tubulin was from Neomarkers (Fremont, CA) and monoclonal antibody against
-tubulin was from Sigma Chemical.
Cell culture and treatments
Chinese hamster V79 cells or human Hela cells (ATCC, Rockville, MD) were grown at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (high glucose) or Iscove (Gibco BRL Products), respectively, supplemented with 5% fetal bovine serum (FBS; Immunocorp Sciences, Montreal, CA) and penicillin (100 U/ml)/streptomycin (100 µg/ml)/ amphotericin B (0.25 µg/ml), pH 7.4. Human primary skin fibroblasts from a normal individual or derived from two HT1 patients (designated HF and WG) were obtained from Dr H.Levy (Harvard Medical School, Boston, MA) and Dr C.R.Scriver (Montreal Children’s Hospital, Montreal, Quebec, Canada) and grown in minimal essential medium (Gibco BRL Products) supplemented with 10% FBS and antibiotics/antimycotic as above. Both HT1 cell lines were negative for immunoreactive FAH. For experiments, cells were initially seeded at a density of 2.5 x 104 cells/ml of medium, which allowed exponential growth at the start of treatment, 24 h after seeding. FAA (100 µM) was added to the medium and treatment was terminated 2 h later. If applicable, cells were left to recover in fresh medium for the indicated times. When the protein kinase inhibitors PD98059 (75 µM), wortmannin (1 µM) or staurosporine (10 nM), or the tyrosine kinase inhibitor genistein (30 µM), the adenylate cyclase activator forskolin (20 µM) or the farnesyltransferase inhibitor B581 (50 µM) were used, cells were preincubated for 1 h with each chemical before FAA treatment. Pre-treatment with GSH-replenishing agents (20 mM GSH-MEE or 1 mM NAC) was for 2 h. The general growth factor receptor inhibitor suramin (300 µM) or the EGFR inhibitor tyrphostin AG1478 (1 µM) were added 5 min before FAA and then left with FAA for 2 h. Cells were harvested at the end of FAA treatment or at the indicated times post-treatment and processed for different analysis (Giemsa staining, immunofluorescence microscopy or western blot) as indicated below.
Cytological analysis by Giemsa staining
Giemsa staining of cells was performed basically as described by Dean and Danford (72). Briefly, following FAA treatment, cells grown on coverslips were rapidly rinsed twice with a mixture of equal volumes of PBS and methanol/acetic acid (3/1). Cells were then fixed in cold methanol/acetic acid (3/1) during 30 min at room temperature and then coverslips were air-dried. Cells were stained with Giemsa dye (Gibco BRL) by immersing coverslips in freshly prepared Giemsa solution (12% v/v in 0.01 M phosphate buffer pH 6.8). Following two washes in phosphate buffer, coverslips were air-dried, mounted and examined in a phase-contrast microscope (Leitz DMRB). A total of 100 mitotic cells or 200 interphasic cells/coverslip were counted.
Western blot analysis
For analysis of phosphorylated ERK1/2 protein levels, cells grown on 6 cm Petri dishes were directly scraped in loading buffer (62.5 mM Tris–HCl pH 6.8, 2.3% SDS, 10% glycerol, 5% ß-mercapthoethanol) at the indicated time post-FAA treatment. For analysis of soluble and polymerized tubulin, FAA-treated cells were lysed in a microtubule-stabilizing buffer containing paclitaxel and free tubulin and microtubule fractions were separated as described by Legault et al. (73). Proteins (10 µg or equivalent to 10 x 104 cells) were separated by SDS–PAGE in a 12% gel. After blotting, nitrocellulose membranes were blocked for 1 h with 1% blocking solution (ECL kit; Boehringer Mannheim, Mannheim, Germany) and probed overnight at 4°C with anti-phosphorylated ERK1/2 (1/1000) or anti-ß-tubulin (1/400). An anti-rabbit or -mouse IgG-horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as secondary conjugated antibody. Signals were revealed using the enhanced chemiluminescence detection system (ECL; Boehringer Mannheim).
Cell synchronization and cell cycle analysis by flow cytometry
V79 and Hela cells grown in T25 flasks or on coverslips were synchronized at the G1/S transition, respectively, by mimosine treatment, as described previously (10), or by a standard double thymidine block protocol (74). Synchronized cells were treated with FAA (100 µM x 2 h) in late S/early G2, which took place at 5.5 h (V79) or 6 h (Hela) after the release from the G1/S blockage. Cells were harvested at different time points after FAA treatment and processed for cell cycle analysis by fluorescence-activated cell sorter (FACS) as described previously (10) or for analysis by immunofluorescence microscopy as described below.
Cell analysis by immunofluorescence microscopy
Coverslips with exponentially growing cells or synchronized cells treated with FAA (pre-treated or not with inhibitors) were rapidly rinsed with PBS, fixed in methanol (–20°C x 20 min) and incubated with the primary antibody, i.e. anti-phosphorylated ERK1/2 (1/250), anti-NuMA (1/75); anti-PCM (1/500), CREST serum (1/500), anti-Golgin-97 (1/40), anti-ß-tubulin (1/500) or anti--tubulin (1/1000) for 1 h. After brief washing with PBS/0.1% Tween 20, cells were incubated with the secondary antibody (anti-rabbit, -mouse or -human IgG coupled to FITC or Texas Red; Jackson ImmunoResearch Laboratories) for 1 h. Cells were counterstained with Hoescht at the final washing step and then coverslips were mounted and examined in a fluorescence microscope (Leitz DMRB). For quantitation of the frequency of CREST-positive micronuclei formation, CREST-labeled micronuclei were scored in 200 cells/coverslip, with two coverslips per treatment.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs J.B.Rattner and M.Fritzler (University of Calgary, Calgary, Canada) for the gift of antibodies against NuMA, PCM, Golgin-97 and CREST serum. We thank Drs J.Landry and J.Huot (Hotel-Dieu Hospital Research Center, Quebec, Canada) for providing us with some specific reagents. We also thank F.Lettre, N.Lamère and J.Marchand (Université Laval, Quebec, Canada) for technical assistance in some specific experiments and M.Dufour (CHUL Research Center, Quebec, Canada) in the analysis by flow cytometry. This work was supported by a grant from the Medical Research Council of Canada to R.M.T. (MT-11086).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Laboratory of Cell and Developmental Genetics, Department of Medicine, Pav. C.-E. Marchand, Université Laval, Sainte-Foy, Quebec, G1K 7P4, Canada. Tel: +1 418 656 3339; Fax: +1 418 656 7176; Email: robert.tanguay@rsvs.ulaval.ca
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Mitchell, G.A., Grompe, M., Lambert, M. and Tanguay, R.M. (2001) Hypertyrosinemia. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York, pp. 1777–1805.
2 Weinberg, A., Mize, C. and Worthen, H. (1976) The occurrence of hepatoma in the chronic form of hereditary tyrosinemia. J. Pediatr., 88, 434–438.[ISI][Medline]
3 Lindblad, B., Lindstedt, S. and Steen, G. (1977) On the enzymic defects in hereditary tyrosinemia. Proc. Natl Acad. Sci. USA, 74, 4641–4645.
4 Phaneuf, D., Lambert, M., Laframboise, R., Mitchell, G., Lettre, F. and Tanguay, R.M. (1992) Type 1 hereditary tyrosinemia. Evidence for molecular heterogeneity and identification of a causal mutation in a French Canadian patient. J. Clin. Invest., 90, 1185–1192.
5 Edwards, S.W. and Knox, W.E. (1955) Homogentisate metabolism: the isomerization of maleylacetoacetate by an enzyme which requires glutathione. J. Biol. Chem., 220, 79–91.
6 Laberge, C., Lescault, A. and Tanguay, R.M. (1986) Hereditary tyrosinemias (type I): a new vista on tyrosine toxicity and cancer. In Poirier, L.A., Newberne, P.M. and Pariza, M.W. (eds), Essential Nutrients in Carcinogenesis. Plenum Press, New York, pp. 209–221.
7 Lloyd, A.J., Gray, R.G.F. and Green, A. (1995) Tyrosinaemia type 1 and glutathione synthetase deficiency: two disorders with reduced hepatic thiol group concentrations and a liver 4-fumarylacetoacetate hydrolase deficiency. J. Inherit. Metab. Dis., 18, 48–55.[ISI][Medline]
8 Tanguay, R.M., Jorquera, R., Poudrier, J. and St-Louis, M. (1996) Tyrosine and its catabolites: from disease to cancer. Acta Biochim. Pol., 43, 209–216.[ISI][Medline]
9 Jorquera, R. and Tanguay, R.M. (1997) The mutagenicity of the tyrosine metabolite, fumarylacetoacetate, is enhanced by glutathione depletion. Biochem. Biophys. Res. Commun., 232, 42–48.[ISI][Medline]
10 Jorquera, R. and Tanguay, R.M. (1999) Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis. FASEB J., 13, 2284–2298.
11 Kubo, S., Sun, M., Miyahara, M., Umeyama, K., Urakami, K., Yamamoto, T., Jakobs, C., Matsuda, I. and Endo, F. (1998) Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetoacetate and is inhibited by caspase inhibitors. Proc. Natl Acad. Sci. USA, 95, 9552–9557.
12 Endo, F., Kubo, S., Awata, H., Kiwaki, K., Katoh, H., Kanegae, Y., Saito, I., Miyazaki, J., Yamamoto, T., Jakobs, C. et al. (1997) Complete rescue of lethal albino c14CoS mice by null mutation of 4-hydroxyphenylpyruvate dioxygenase and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of the tyrosine catabolic pathway. J. Biol. Chem., 272, 24426–24432.
13 Gilbert-Barness, E., Barness, L.A. and Meisner, L.F. (1990) Chromosomal instability in hereditary tyrosinemia type I. Pediatr. Pathol., 10, 243–252.[Medline]
14 Zerbini, C., Weinberg, D.S., Hollister, K.A. and Perez-Atayde, A.R. (1992) DNA ploidy abnormalities in the liver of children with hereditary tyrosinemia type I. Correlation with histopathologic features. Am. J. Pathol., 140, 1111–1119.[Abstract]
15 Onfelt, A. (1986) Mechanistic aspects on chemical induction of spindle disturbances and abnormal chromosome numbers. Mutat. Res., 168, 249–300.[ISI][Medline]
16 Parry, J.M., Parry, E.M., Bourner, R., Doherty, A., Ellard, S., O’Donovan, J., Hoebee, B., de Stoppelaar, J.M., Mohn, G.R., Onfelt, A. et al. (1996) The detection and evaluation of aneugenic chemicals. Mutat. Res., 353, 11–46.[ISI][Medline]
17 Sen, C.K. (2000) Cellular thiols and redox-regulated signal transduction. Curr. Top. Cell. Regul., 36, 1–30.[ISI][Medline]
18 Knebel, A., Rahmsdorf, H.J., Ullrich, A. and Herrlich, P. (1996) Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents. EMBO J., 15, 5314–5325.[ISI][Medline]
19 Cavigelli, M., Li, W.W., Lin, A., Su, B., Yoshioka, K. and Karin, M. (1996) The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J., 15, 6269–6279.[ISI][Medline]
20 Pages, G., Lenormand, P., L’Allemain, G., Chambard, J.C., Meloche, S. and Pouyssegur, J. (1993) Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc. Natl Acad. Sci. USA, 90, 8319–8323.
21 Hayne, C., Tzivion, G. and Luo, Z. (2000) Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole. J. Biol. Chem., 275, 31876–31882.
22 Lewis, T.S., Shapiro, P.S. and Ahn, N.G. (1998) Signal transduction through MAP kinase cascades. Adv. Cancer Res., 74, 49–139.[ISI][Medline]
23 Cobb, M.H. and Goldsmith, E.J. (1995) How MAP kinases are regulated. J. Biol. Chem., 270, 14843–14846.
24 Denhardt, D.T. (1996) Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem. J., 318, 729–747.
25 Gotoh, Y., Nishida, E. and Sakai, H. (1990) Okadaic acid activates microtubule-associated protein kinase in quiescent fibroblastic cells. Eur. J. Biochem., 193, 671–674.[ISI][Medline]
26 Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D. and Rapp, U.R. (1993) Protein kinase C activates RAF-1 by direct phosphorylation. Nature, 364, 249–252.[Medline]
27 Cowley, S., Paterson, H., Kemp, P. and Marshall, C.J. (1994) Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and transformation of NIH 3T3 cells. Cell, 77, 841–852.[ISI][Medline]
28 Mansour, S.J., Matten, W.T., Hermann, A.S., Candia, J.M., Rong, S., Fukasawa, K., Vande Houde, G.F. and Ahn, N.G. (1994) Transformation of mammalian cells by constitutively active MAP kinase kinase. Science, 265, 966–970.
29 Schmidt, C.M., McKillop, I.H., Cahill, P.A. and Sitzmann, J.V. (1997) Increased MAPK expression and activity in primary human hepatocellular carcinoma. Biochem. Biophys. Res. Commun., 236, 54–58.[ISI][Medline]
30 Saavedra, H.I., Fukasawa, K., Conn, C.W. and Stambrook, P.J. (1999) MAPK mediates RAS-induced chromosome instability. J. Biol. Chem., 274, 38083–38090.
31 Fukasawa, K. and Vande Woude, G.F. (1995) Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle. Proc. Natl Acad. Sci. USA, 92, 3430–3434.
32 Thyberg, J. and Moskalewski, S. (1999) Role of microtubules in the organization of the Golgi complex. Exp. Cell Res., 246, 263–279.[ISI][Medline]
33 Griffith, K.J., Chan, E.K., Lung, C.C., Hamel, J.C., Guo, X., Miyachi, K. and Fritzler, M.J. (1997) Molecular cloning of a novel 97-kd Golgi complex autoantigen associated with Sjogren’s syndrome. Arthritis Rheum., 40, 1693–1702.[ISI][Medline]
34 Brule, S., Rabahi, F., Faure, R., Beckers, J.F., Silversides, D.W. and Lussier, J.G. (2000) Vacuolar system-associated protein-60: a protein characterized from bovine granulosa and luteal cells that is associated with intracellular vesicles and related to human 80K-H and murine ß-glucosidase II. Biol. Reprod., 62, 642–654.
35 Fukasawa, K. and Vande Woude, G.F. (1997) Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability. Mol. Cell. Biol., 17, 506–518.[Abstract]
36 Babia, T., Ayala, I., Valderrama, F., Mato, E., Bosch, M., Santaren, J.F., Renau-Piqueras, J., Kok, J.W., Thomson, T.M. and Egea, G. (1999) N-Ras induces alterations in Golgi complex architecture and in constitutive protein transport. J. Cell Sci., 112, 477–489.[Abstract]
37 Russo, T., Zambrano, N., Esposito, F., Ammendola, R., Cimino, F., Fiscella, M., Jackman, J., O’Connor, P.M., Anderson, C.W. and Appella, E. (1995) A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress. J. Biol. Chem., 270, 29386–29391.
38 Denko, N.C., Giaccia, A.J., Stringer, J.R. and Stambrook, P.J. (1994) The human Ha-ras oncogene induces genomic instability in murine fibroblasts within one cell cycle. Proc. Natl Acad. Sci. USA, 91, 5124–5128.
39 Onfelt, A. (1987) Spindle disturbances in mammalian cells. IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism. Mutat. Res., 182, 155–172.[ISI][Medline]
40 Rodriguez-Viciana, P., Warne, P.H., Khwaja, A., Marte, B.M., Pappin, D., Das, P., Waterfield, M.D., Ridley, A. and Downard, J. (1997) Role of PI3K in cell transformation and control of the actin cytoskeleton by ras. Cell, 89, 457–467.[ISI][Medline]
41 Agapova, L.S., Ivanov, A.V., Sablina, A.A., Kopnin, P.B., Sokova, O.I., Chumakov, P.M. and Kopnin, B.P. (1999) p53-dependent effects of RAS oncogene on chromosome stability and cell cycle checkpoints. Oncogene, 18, 3135–3142.[ISI][Medline]
42 Aoshiba, K., Yasui, S., Nishimura, K. and Nagai, A. (1999) Thiol depletion induces apoptosis in cultured lung fibroblasts. Am. J. Respir. Cell Mol. Biol., 21, 54–64.
43 Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T. and Saltiel, A.R. (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem., 270, 27489–27494.
44 Hawes, B.E., van Biesen, T., Koch, W.J., Luttrell, L.M. and Lefkowitz, R.J. (1995) Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J. Biol. Chem., 270, 17148–17153.
45 van Biesen, T., Hawes, B.E., Luttrell, D.K., Krueger, K.M., Touhara, K., Porfiri, E., Sakaue, M., Luttrell, L.M. and Lefkowitz, R.J. (1995) Receptor-tyrosine-kinase- and Gß-mediated MAP kinase activation by a common signalling pathway. Nature, 376, 781–784.[Medline]
46 Yu, R., Lei, W., Mandlekar, S., Weber, M.J., Der, C.J., Wu, J. and Kong, A.T. (1999) Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. J. Biol. Chem., 274, 27545–27552.
47 Chen, W., Martindale, J.L., Holbrook, N.J. and Liu, Y. (1998) Tumor promoter arsenite activates extracellular signal-regulated kinase through a signaling pathway mediated by epidermal growth factor receptor and Shc. Mol. Cell. Biol., 18, 5178–5188.
48 Wang, D., Yu, X., Cohen, R.A. and Brecher, P. (2000) Distinct effects of N-acetylcysteine and nitric oxide on angiotensin II-induced epidermal growth factor receptor phosphorylation and intracellular Ca2+ levels. J. Biol. Chem., 275, 12223–12230.
49 Renglin, A., Harmala-Brasken, A.S., Eriksson, J.E. and Onfelt, A. (1999) Mitotic aberrations induced by carbaryl reflect tyrosine kinase inhibition with coincident up-regulation of serine/threonine protein phosphatase activity: implications for coordination of karyokinesis and cytokinesis. Mutagenesis, 14, 327–333.