HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/13/2539 01-0360fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by SAUER, H. Articles by WARTENBERG, M. Search for Related Content PubMed PubMed Citation Articles by SAUER, H. Articles by WARTENBERG, M.

Activation of p90RSK and growth stimulation of multicellular tumor spheroids are dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP ¹

Department of Neurophysiology, University of Cologne, D-50931 Cologne, Germany

²Correspondence: Department of Neurophysiology, University of Cologne, Robert-Koch-Str. 39, D-50931 Cologne, Germany. E-mail: mw{at}physiologie.uni-koeln.de

SPECIFIC AIMS

Purinergic receptor agonists (e.g., ATP) exert mitogenic effects on tumor cells. The aim of the present study was to investigate the signaling pathways underlying ATP-mediated growth stimulation of multicellular tumor spheroids and the role of reactive oxygen species (ROS) as second messengers within the signal transduction cascade.

PRINCIPAL FINDINGS

1. Generation of ROS and growth stimulation of multicellular prostate tumor spheroids on purinergic receptor stimulation
Incubation of multicellular prostate tumor spheroids with ATP (1 µM, 10 µM, 100 µM) dose-dependently stimulated tumor growth, the most pronounced effect being achieved with 10 µM ATP (Fig. 1 A). Using the redox-sensitive fluorescence indicator 2'7'-dichlorodihydrofluorescein diacetate (H₂DCFDA), a significant elevation of ROS was observed within 60–180 s after treatment of tumor spheroids with ATP and purinergic receptor agonists UTP, ADP, 2-methyl-thio-ATP (2-MeS-ATP), and ADP. ATP-induced growth stimulation was completely inhibited in the presence of the free radical scavengers vitamin E, N-acetyl cysteine (NAC), and dimethyl thiourea (DMTU), indicating a role of the intracellular redox state for the observed growth effect (Fig. 1B ). Elevation of the intracellular redox state was abolished in the presence of the purinergic receptor antagonist suramin, suggesting that ROS generation after treatment of tumor spheroids with nucleotides requires purinergic receptor stimulation.

View larger version (18K): [in this window] [in a new window]   Figure 1. Redox-sensitive growth stimulation of multicellular prostate tumor spheroids. A) Growth stimulation of multicellular tumor spheroids was achieved by treatment with 1 µM, 10 µM, and 100 µM ATP. The tumor spheroids were treated from day 3 to day 6 of spheroid culture with ATP. Cell culture medium was completely exchanged every 24 h. B) ATP-induced growth stimulation was abolished by coadministration of the free radical scavengers NAC (20 mM), DMTU (1 mM), vitamin E (30 µM), and the MEK1/2 inhibitor PD98059. Tumor spheroids were treated with 10 µM ATP in either the presence or absence of free radical scavengers and PD98059. The size of tumor spheroids was evaluated after 24 h. *P < 0.05, significantly different from the ATP-treated sample.

2. A nonphagocytic NADPH oxidase regulated by Ca²⁺-dependent phospholipase A₂ (PLA₂) is the source of ATP-induced ROS
To evaluate the source of ROS generated after purinergic receptor stimulation by ATP, tumor spheroids were incubated with the NADPH-oxidase inhibitors diphenylene iodonium chloride (DPI) and 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), which significantly inhibited the ATP-evoked ROS response. ROS generation by ATP was dependent on ATP-induced transient elevation of the intracellular calcium concentration [Ca²⁺]_i and the activity of Ca²⁺-dependent PLA₂, since ROS generation was abolished upon chelation of intracellular Ca²⁺ and coadministration of the PLA₂ antagonists indomethacin and methyl arachidonyl fluorophosphonate.

3. Activation of the extracellular signal-regulated kinase (ERK1/2) mitogen-activated protein kinase (MAPK) pathway by ATP is not dependent on elevation of intracellular ROS and intracellular Ca²⁺
Incubation with the MEK1/2 inhibitor PD98059 inhibited the growth stimulation of multicellular tumor spheroids by ATP (see Fig. 1B ), indicating an involvement of the ERK1/2 MAPK pathway. Activation of the ERK1/2 pathway may be regulated by ROS and by Ca²⁺ acting as second messenger. As evaluated by the use of phospho-specific antibodies and immunohistochemistry, ERK1/2 was transiently phosphorylated after treatment of tumor spheroids with ATP, the maximum activation being observed after 5 min. ERK1/2 activation was not impaired in the presence of the free radical scavengers vitamin E, NAC, and DMTU or under conditions where intracellular Ca²⁺ was chelated. This clearly indicates that ERK1/2 activation and activation of upstream members of the ERK1/2 MAPK pathway are not dependent on the elevation of ROS after purinergic receptor stimulation.

4. Activation of p90RSK after purinergic receptor stimulation by ATP requires elevation of ROS and phosphorylation of ERK1/2
The downstream effector of ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which, like ERK1/2, is phosphorylated upon activation and translocated to the cell nucleus. Treatment of tumor spheroids with ATP resulted in a transient phosphorylation of p90RSK, with maximum activation after 5 min. Activation of p90RSK by ATP was inhibited upon coadministration of PD98059, suggesting that the ATP-evoked p90RSK activation is transduced via the ERK1/2 pathway. In contrast to ERK1/2, preincubation with either the radical scavenger vitamin E, NAC, or DMTU totally inhibited activation by ATP (Fig. 2 ), indicating that p90RSK downstream of ERK1/2 is the molecular target for ATP-induced ROS.

View larger version (47K): [in this window] [in a new window]   Figure 2. Inhibition of the ATP-induced activation of p90RSK by free radical scavengers. A) p90RSK immunofluorescence representative in either untreated (control) whole mount tumor spheroids (Aa) or tumor spheroids treated with 10 µM ATP (Ab), 10 µM ATP + 20 mM NAC (Ac), 10 µM ATP + 1 mM DMTU (Ad), and 10 µM ATP + 30 µM vitamin E (Ad). B) Quantitative presentation of the p90RSK immunofluorescence after treatment of multicellular tumor spheroids for different times with either ATP alone or ATP in the presence of free radical scavengers. The bar represents 15 µm. *P < 0.05, significantly different from the ATP-treated sample.

CONCLUSIONS

Although a variety of purinergic receptor subtypes have been described, a precise investigation of the signal transduction cascades activated by purinergic receptor stimulation is still lacking. Purinergic receptor agonists have been shown to exert mitogenic effects in numerous preparations. Stimulation of cell growth has likewise been reported for low concentrations of ROS. These observations led us to hypothesize that purinergic receptor stimulation may increase intracellular ROS, which could act as signaling molecules in the signal transduction cascade elicited by purinergic receptor agonists. In a previous study, we reported that ATP is actively secreted upon mechanical stimulation of prostate cancer cells. ATP released from prostate cells has been discussed to be involved in the autocrine and paracrine regulation of mucus secretion, and (possibly as a side effect) may induce benign as well as neoplastic growth of the prostate tissue.

It is shown for the first time that purinergic receptor agonists elevate intracellular ROS, which are necessary to promote tumor spheroid growth since preincubation with free radical scavengers abolished the observed effect. The elevation of ROS by ATP occurred within a few minutes, which is the period required to activate signal transduction cascades, e.g., MAPK pathways.

ROS may be intracellularly generated by several sources, including mitochondria and a nonphagocytic NADPH oxidase. In contrast to the NADPH oxidase present in phagocytic cells that is used during the ‘oxidative burst’ to defend against invading microorganisms, nonphagocytic NADPH oxidase may evoke a ‘small oxidative burst’ to generate ROS that are acting as signaling molecules in growth factor- and cytokine-induced signaling cascades. The data of the present study strongly suggest that ATP-induced ROS generation occurs via activation of a nonphagocytic NADPH oxidase that we have demonstrated to be present in multicellular prostate tumor spheroids. The activity of the nonphagocytic NADPH oxidase was apparently dependent on the transient elevation of intracellular [Ca²⁺]_i achieved upon treatment of tumor spheroids with purinergic receptor agonists. Furthermore, ROS generation was completely abolished in the presence of inhibitors of Ca²⁺-dependent PLA₂, suggesting that elevation of [Ca²⁺]_i may be necessary for PLA₂ activity, which (comparable to the phagocytic NADPH oxidase of neutrophil cells) induces the assembly of the NADPH oxidase subunits at the plasma membrane.

The ROS generated by the nonphagocytic NADPH oxidase may interfere with signaling cascades activated upon purinergic receptor stimulation. It is demonstrated that the ERK1/2 MAPK pathway was rapidly activated upon treatment of tumor spheroids with ATP, whereas JNK and the p38 MAPK pathway were unaffected (data not shown). ERK1/2 phosphorylation and activation of the signaling cascade upstream of ERK1/2 did not require ROS generated at purinergic receptor stimulation, since ERK1/2 activation was robustly observed in the presence of different free radical scavengers and upon chelation of intracellular Ca²⁺. Under these conditions, the elevation of intracellular ROS by ATP was abolished. The downstream effector of ERK1/2 is p90RSK, which plays an important role in cell growth by activating several transcription factors and the Na⁺/H⁺ exchanger. Activation of p90RSK by exog-enously added H₂O₂ has recently been reported, indicating a possible redox regulation of the activity of this protein kinase. Our data show that in contrast to ERK1/2, p90RSK phosphorylation was blunted upon coadministration of ATP with free radical scavengers, which clearly indicates that the ROS generated by a nonphagocytic NADPH oxidase are targeting p90RSK downstream of ERK1/2. Phosphorylation p90RSK was abolished by PD98059, which inhibits ERK1/2 activation via MEK1/2. Hence, our data demonstrate that p90RSK phosphorylation after purinergic receptor stimulation requires the generation of intracellular ROS by a nonphagocytic NADPH oxidase and ERK1/2 phosphorylation, since in the absence of either ROS elevation or phosphorylation of ERK1/2, ATP failed to activate p90RSK and stimulate the growth of multicellular tumor spheroids (Fig. 3 ).

View larger version (19K): [in this window] [in a new window]   Figure 3. Regulation of p90RSK by ROS and ERK1/2. A) Activation of p90RSK and growth stimulation of multicellular tumor spheroids requires generation of ROS by a NADPH oxidase after purinergic receptor stimulation as well as the activity of ERK1/2. B) In the absence of ROS, ATP still activates ERK1/2, but p90RSK activation and growth stimulation are abolished. C) Inhibition of ERK1/2 by PD98059 blunts p90RSK activation and growth stimulation despite the presence of elevated ROS after treatment of tumor spheroids with ATP. Activation (+); the absence of activity (-).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0360fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.01-0360fje

This Article Full Text (PDF) All Versions of this Article: 15/13/2539 01-0360fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by SAUER, H. Articles by WARTENBERG, M. Search for Related Content PubMed PubMed Citation Articles by SAUER, H. Articles by WARTENBERG, M.