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Molecular Endocrinology, doi:10.1210/me.2003-0190
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Molecular Endocrinology 18 (1): 162-172
Copyright © 2004 by The Endocrine Society

Nerve Growth Factor Restores p53 Function in Pituitary Tumor Cell Lines via trkA-Mediated Activation of Phosphatidylinositol 3-Kinase

Marco Facchetti, Daniela Uberti, Maurizio Memo and Cristina Missale

Division of Pharmacology, Department of Biomedical Sciences and Biotechnology and Centre of Excellence on Diagnostic and Therapeutic Innovation, University of Brescia, 25123 Brescia, Italy

Address all correspondence and requests for reprints to: Cristina Missale, Ph.D., Department of Biomedical Sciences and Biotechnology, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. E-mail: cmissale{at}med.unibs.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two groups of prolactinomas were identified, one slowly proliferating and responsive to bromocriptine and one fast proliferating and bromocriptine resistant. Nerve growth factor (NGF) inhibits proliferation of bromocriptine-resistant cells by mechanisms that are still unclear.

The tumor suppressor p53 is one of the key regulators of cell proliferation and in most tumors, but not pituitary adenomas, it is inactivated by genomic mutations. Here we investigated whether in prolactinoma cell lines NGF influences cell cycle-related pathways involving p53. By using conformation-specific antibodies and immunocytochemistry we found that in bromocriptine-resistant cells p53 adopts a mutant conformation that precludes its nuclear translocation and transcriptional activity. NGF administration to these cells refolds p53 into wild-type tertiary structure, promotes its nuclear translocation, and restores its DNA-binding activity as demonstrated by the transcriptional activation of p21Cip1/WAF1 and the resulting down-regulation of different cyclins and cyclin-dependent kinase 2. Inactivation of trkA, but not of p75NTR, and wortmannin prevented NGF-induced p53 nuclear translocation. Thus, in prolactinoma cells p53 is inactivated by conformational mutation and cytoplasmic segregation. This defect is reversible because NGF reconstitutes active p53 in these cells. This effect of NGF is exclusively mediated by trkA through activation of phosphatidylinositol-3-kinase and may be related to its growth-inhibitory action.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROLACTINOMAS ARE THE most frequently occurring adenomas in the human pituitary. In previous studies we have developed and characterized two phenotypically different groups of human prolactinoma cell lines (1, 2). Those derived from tumors refractory to the dopaminomimetic therapy (NR) not only lack D2 receptors, but also have a high proliferation rate in vitro and high tumorigenic potential in vivo, whereas those derived from the dopamine-sensitive prolactinomas (R) proliferate slowly and lack tumorigenicity (1). Proliferation of R cells, as well as the expression of D2 receptors, is under the control of an autocrine loop mediated by nerve growth factor (NGF) that is disrupted in NR cells (2). NGF administration to NR not only induces D2 receptor expression, but also remarkably inhibits the proliferation rate and abrogates the anchorage-independent clonal growth in soft agar and the tumorigenic potential in vivo of these cells (1, 3). The molecular mechanisms underlying the latter effects are still elusive.

NGF interacts with trkA and p75NTR receptors (4). The tyrosine kinase trkA signals through a Ras-dependent pathway ending with activation of the mitogen-activated phosphokinases (Erk) (5) and through other enzymes, such as phosphatidylinositol 3-kinase (PI3K) (6) and phospholipase C{gamma} (PLC{gamma}) (7), whereas p75NTR stimulates ceramide production and activates both nuclear factor-{kappa}B (NF-{kappa}B) and c-Jun N-terminal kinase (8). How these receptors act, individually or together, to regulate specific cell responses to NGF and the nature of the intracellular signals activated are key questions in NGF signal transduction. We have recently reported that, in prolactinoma cells, D2 receptor expression is controlled by NGF via binding to p75NTR and activation of NF-{kappa}B, in a trkA-independent way (9). However, the receptor subtypes and intracellular pathways involved in NGF-mediated growth arrest of these cells have not been identified so far. The role of trkA and p75NTR in cell growth control is still controversial. In particular, trkA has been shown to promote proliferation in some tumor cell lines (10, 11) but to induce cell growth inhibition in others (12, 13, 14). Similarly, it has been reported that p75NTR is a tumor suppressor in certain tumors (15, 16) but is mitogenic in others (17). Therefore, identifying the mechanisms involved in NGF-mediated cell growth inhibition in prolactinoma cells may help to clarify this issue.

Cell proliferation is regulated at specific check points in the cell cycle (18). In particular, an important restriction point in the G1 phase, which is controlled by the tumor suppressor protein p53 and its effector p21Cip1/WAF1, has been identified (18). The importance of this mechanism is demonstrated by the fact that inactivation of this pathway is related to tumorigenesis. More than 50% of all human cancers show inactivating mutations of the p53 gene (19) and in cells that retain wild-type p53, other defects in this pathway have been identified. In particular, changes in the tertiary structure from a wild-type to a mutant conformation (20, 21) and aberrant cytoplasmic sequestration are emerging as important mechanisms of p53 inactivation (22, 23, 24, 25, 26). Studies on pituitary tumors, in contrast to most of solid tumors, have failed to identify mutations in the p53 gene (27, 28, 29). These findings, together with the observation that p53 is implicated in NGF-induced growth arrest of neuronal cells (30, 31), prompted us to test the hypothesis that p53 might mediate the antiproliferative action of NGF in prolactinoma cells.

We found that in the more transformed NR cells, but not in the more differentiated R phenotype, p53 adopts a mutant conformation that precludes its nuclear localization and transcriptional activity. This defect is reversible because NGF administration to these cells refolds p53 into the wild-type conformation, promotes its nuclear translocation, and restores its transcriptional activity. This effect, resulting in the expression of p21Cip1/WAF1 and subsequent down-regulation of different cyclins and cyclin-dependent kinases (cdks), is mediated by trkA-induced activation of the PI3K-Akt pathway in a p75NTR-independent way.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression, Conformation, and Cellular Localization of p53 in Prolactinoma Cells
The expression of p53 was evaluated by both RT-PCR and Western blot (WB) in two prolactinoma cell lines, one R, characterized by low proliferation rate and NGF production, and one NR, which has high proliferation rate and lacks NGF production. RT-PCR analysis revealed that p53 mRNA was present in both R and NR cell lines at a similar expression level (Fig. 1AGo, lanes 1 and 2) and that exposure of NR cells to 100 ng/ml NGF for 5 d did not substantially modify p53 mRNA expression (Fig. 1AGo, lane 3). Direct amplification of the RNA, i.e. omitting the reverse transcription reaction, did not produce any band (data not shown), confirming the specificity of the PCR. Similar results were obtained by WB with the specific anti-p53 antibody that recognizes both wild-type and mutated p53 forms. A representative WB is reported in Fig. 1BGo and the densitometric analysis of three independent blots, with p53 signals normalized to the corresponding ß-tubulin staining, is reported in Fig. 1CGo. R and NR cells exhibited comparable p53 levels, and treatment of NR cells with NGF did not modify p53 concentration.



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  		Fig. 1. Expression of p53 in Prolactinoma Cells

A, p53 mRNA levels in R and untreated or NGF-treated NR cells. The cDNA was PCR amplified (28 cycles) with specific primers as described in Materials and Methods. The amount of cDNA in each sample was determined by PCR amplification of ß-actin. B, Representative WB analysis of p53 protein in R, NR, and NGF-treated NR cells. Aliquots of cell proteins (15 µg/lane) were resolved by 10% SDS-PAGE and immunoreacted with the anti-p53 antibody as described in Materials and Methods. The amount of proteins in each lane was evaluated by immunoreaction with the ß-tubulin antibody. C, Densitometric analysis of p53 normalized to the corresponding ß-tubulin levels. Bars represent the mean ± SE of three experiments.

 
There is now increasing evidence that one of the intrinsic properties of p53 is conformational flexibility that causes it to adopt at least two tertiary structures in vivo, a wild-type functionally active and a mutant functionally inactive conformation (32). The conformational status of p53 in R and NR prolactinoma cell lines was evaluated by immunoprecipitation with conformation-specific monoclonal antibodies that allow discriminating folded vs. unfolded p53. In particular, the wild-type form is reactive with the PAb1620 antibody, which binds to a denaturation-sensitive epitope within the DNA-binding domain, whereas the so-called "mutant" form reacts with PAb240 antibody recognizing a primary epitope that is cryptic in the wild-type conformation (32, 33). Immunoprecipitated p53 was detected by WB with the polyclonal antibody CM-1. As shown in the representative immunoprecipitation reported in Fig. 2Go, in R cells p53 had high reactivity with PAb1620 (lane 3), but not with PAb240 (lane 2), suggesting that in this cell line p53 adopts its wild-type conformation. By contrast, in NR cells, p53 showed reactivity with PAb240 (lane 2), with virtually no interaction with PAb1620 (lane 3), suggesting that in this cell line p53 takes on an immunological phenotype identical to that of many of its mutated forms. Interestingly, exposure of NR cells to NGF resulted in p53 folding into the wild-type conformation as shown by increased reactivity with PAb1620 (lane 3). Data obtained in three independent experiments were analyzed by densitometry and expressed as the ratio between PAb1620- and PAb240-immunoprecipitated p53 (1620/240). As reported in Fig. 2BGo, the 1620/240 ratio was high in R cells and very low in NR, indicating the prevalence of wild-type and mutant p53 forms, respectively. NGF induced a 9-fold increase in the 1620/240 ratio in NR cells, indicating a relevant increase of wild-type p53 over its mutant form. In Fig. 2AGo it also appears that NGF treatment increased the precipitation of p53 with PAb1801 (lane 1), which recognizes an epitope localized in p53 N-terminal domain and exposed in both wild-type and mutant protein forms. In line with this observation it has been reported that zinc treatment, which refolded metal chelator-induced mutant-like p53 into the wild-type conformation, also increased precipitation of p53 with both DO7 (reacting with an epitope in the N terminus) and PAb421 (reacting with an epitope in the C terminus) antibodies (34), suggesting that the conformational change induced by treatments likely increases the accessibility of p53 extremities to the antibodies.



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  		Fig. 2. Analysis of p53 Protein Conformation in Prolactinoma Cells

A, Cell lysates from R, NR, and NGF-treated NR cell lines were immunoprecipitated (IP) with the monoclonal antibodies PAb1620 (wild type specific), PAb240 (mutant specific), and PAb1801 (both p53 conformations). Immunoprecipitated p53 was detected by WB with the polyclonal anti-p53 antibody CM-1. A representative WB is shown. B, Data were analyzed by densitometry and expressed as the ratio between PAb1620- and PAb240-immunoprecipitated p53. Bars represent the mean ± SE of three independent experiments. *, P < 0.001 vs. untreated NR cells, Student’s t test.

 
The subcellular localization of p53 in R and NR cells was analyzed by immunocytochemistry with a monoclonal antibody interacting with both wild-type and mutant p53. As shown in Fig. 3Go, in R cells, where p53 adopted its wild-type conformation, p53 immunoreactivity was present in both the nucleus and the cytoplasm (Fig. 3AGo, panel a). In particular, a prevalent nuclear localization of p53 was detected in about 20% of cells (panel d) and a diffuse nuclear and cytoplasmic localization in the remaining cells (panel e). In NR cells, where the preferential p53 conformation was mutant, p53 immunoreactivity was localized mainly in the cytoplasm with only a faint nuclear staining (Fig. 3AGo, panels b and f), suggesting that in this cell phenotype mutant p53 was cytoplasmically segregated. Thus, in prolactinoma cell lines there is a close correlation between p53 conformation and subcellular localization. As depicted in panel c, administration of NGF (100 ng/ml; 5 d) to NR cells relieved p53 cytoplasmic sequestration and promoted its nuclear translocation. In particular, in about 30% of NGF-treated NR cells p53 immunoreactivity was more concentrated in the nucleus than in the cytoplasm (panel g), and in the remaining cells it was equally distributed between these two compartments (panel h). This observation was further supported by WB on nuclear and cytoplasmic proteins from NR cells. As shown in Fig. 3Go, B and C, similar p53-immunoreactive bands were detectable in the cytosolic fractions from untreated and NGF-treated NR cells. By contrast, in the nuclear fraction from untreated NR cells the p53-immunoreactive band was hardly detectable, and its intensity was increased by NGF treatment (Fig. 3Go, D and E). Taken together, these data suggest that in human prolactinoma cell lines the conformation and cellular distribution of p53 are related to the degree of transformation and are dependent on the NGF system.



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  		Fig. 3. Subcellular Distribution of p53 in Prolactinoma Cells

A, R, NR, and NGF-treated NR cells were fixed with methanol at -20 C and processed for p53 immunostaining as described in Materials and Methods. Panel a, Distribution of p53 immunoreactivity in R cells detected at x40 magnification; panels d and e, representative R cells detected at x100 magnification showing that in some cells p53 staining is concentrated into the nucleus (d) and in others it is equally distributed between the nucleus and the cytoplasm (e); panel b, distribution of p53 immunoreactivity in NR cells detected at x40 magnification; panel f, representative NR cells detected at x100 magnification showing that p53 immunostaining is segregated into the cytoplasm; panel c, distribution of p53 immunoreactivity in NGF-treated NR cells (x40 magnification); panels g and h, representative NGF-treated NR cells detected at x100 magnification showing that in some cells p53 immunostaining is preferentially localized into the nucleus (g), whereas in others (h) it is equally distributed between the nucleus and the cytoplasm. Scale bar, 100 µm. B, Representative WB analysis of p53 in cytoplasmic extracts from untreated and NGF-treated NR cells. Aliquots of proteins (15 µg/lane) were analyzed for p53 and ß-tubulin levels as described in Materials and Methods. C, Densitometric analysis of cytoplasmic p53 normalized to the corresponding ß-tubulin levels. Bars represent the mean ± SE of three experiments. D, Representative WB analysis of p53 in nuclear extracts from untreated and NGF-treated NR cells. Aliquots of proteins (30 µg/lane) were analyzed for p53 and ß-actin levels as described in Materials and Methods. E, Densitometric analysis of nuclear p53 normalized to the corresponding ß-actin levels. Bars represent the mean ± SE of three experiments. *, P < 0.001 vs. untreated cells, Student’s t test.

 
NGF Activates the p53/p21Cip1/WAF1 Pathway in NR Prolactinoma Cells
To prove that p53 conformational folding and nuclear translocation induced by NGF in NR cells have a functional significance, we measured the expression of p21Cip1/WAF1 and Mdm2, two p53 target genes. The expression of p21Cip1/WAF1 was evaluated by WB and that of Mdm2 by RT-PCR. As shown in Fig. 4Go, A and B, the levels of expression of p21Cip1/WAF1, which were very low in untreated NR cells, were significantly increased in response to NGF treatment (100 ng/ml, 5 d), suggesting that in prolactinoma cells the p53/p21Cip1/WAF1 pathway was efficiently activated by NGF. Similarly, NGF administration to NR cells induced a significant increase in the expression of Mdm2 mRNA (Fig. 4CGo).



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  		Fig. 4. NGF Induces the Expression of p21Cip1/WAF1 and Down-Regulates Different Cyclins and cdk2 in NR Prolactinoma Cells

A, Effect of NGF on p21Cip1/WAF1 expression. Proteins (30 µg/lane) from untreated and NGF-treated NR cells were analyzed by WB with the anti-p21Cip1/WAF1 antibody. To ensure equal protein loading, membranes were stripped and reprobed with the anti-ß-tubulin antibody. B, Densitometric analysis of p21Cip1/WAF1 normalized to the corresponding ß-tubulin levels. Bars represent the mean ± SE of three experiments. *, P < 0.001 vs. untreated cells, Student’s t test. C, Effect of NGF treatment on Mdm2 mRNA levels. The amount of cDNA in each sample was checked by PCR amplification with ß-actin primers. Data are representative of three experiments. D, RT-PCR analysis of cyclin A mRNA levels in untreated and NGF-treated NR cells. Data are representative of three experiments. E, Representative WB showing the effect of NGF on cyclin E and cdk2 expression. F and G, Densitometric analysis of cyclin E (F) and cdk2 (G) normalized to the corresponding ß-tubulin levels. Bars represent the mean ± SE of three experiments. *, P < 0.001 vs. untreated cells, Student’s t test.

 
Because p21Cip1/WAF1 acts as an inhibitor of different cyclins and cdks to initiate G1 arrest, we measured the expression of cell cycle-related proteins in untreated and NGF-treated NR cells. The expression of cyclin E and cdk2 was evaluated by WB, whereas cyclin A, which was hardly detectable by WB (data not shown), was measured by RT-PCR. A representative WB is reported in Fig. 4EGo. Densitometric analysis of three experiments with cyclin E and cdk2 normalized to the corresponding values of ß-tubulin is shown in Fig. 4Go, F and G. Exposure of NR cells to NGF resulted in a 42% ± 1.7% decrease of cyclin E expression (Fig. 4FGo) and a 62% ± 1.4% decrease of cdk2 expression (Fig. 4GGo). Similarly, as a result of NGF administration, cyclin A mRNA was significantly decreased (Fig. 4DGo). Thus, according to its antiproliferative effects and as a result of activation of the p53/p21Cip1/WAF1 pathway, in NR cells NGF impairs the formation of cyclin A-cdk2 and cyclin E-cdk2 complexes that are essential for cell cycle progression at the G1/S transition (18).

NGF-Induced p53 Nuclear Translocation Is Mediated by trkA-Dependent Activation of PI3K
To evaluate the contribution of trkA and p75NTR receptors to NGF-induced p53 nuclear translocation, each receptor was individually inactivated, and p53 subcellular localization was investigated by immunocytochemistry. On the basis of previous studies (9), p75NTR-mediated responses were inhibited using a specific anti-p75NTR antibody at the concentration of 100 ng/ml, and trkA was inactivated by inhibiting its intrinsic tyrosine kinase activity with 1 µM genistein. As illustrated in Fig. 5BGo, genistein prevented NGF-induced trkA phosphorylation at Tyr490. The ratio ptrkA (max OD)/ß-tubulin (max OD) was 0.13 ± 0.01 (mean ± SE; n = 3) in NGF-treated cells and 0.052 ± 0.02 (mean ± SE; n = 3) in cells exposed to NGF in the presence of genistein. Genistein also abolished the effects of NGF on p53 trafficking. A representative immunocytochemistry is depicted in Fig. 5AGo. As in untreated NR cells (Fig. 5AGo, panels a and e), also in NR cells exposed to NGF in the presence of 1 µM genistein, p53 immunoreactivity was confined to the cytoplasm (panels d and h). By contrast, p53 nuclear translocation induced by NGF in NR cells was not modified by inhibition of p75NTR signaling, as shown by the specific p53 immunostaining that, as in cells exposed to NGF (panels b and f), also in cells exposed to NGF in the presence of the anti-p75NTR antibody, was mainly localized into the nucleus (panels c and g). In these conditions the anti-p75NTR antibody completely prevented NGF-induced NF-{kappa}B activation (Fig. 5CGo), indicating that it efficiently blocked p75NTR-mediated effects. To further demonstrate the role of NGF-mediated trkA activation in the regulation of p53 trafficking, NGF-secreting R cells were either deprived of secreted NGF with an anti-NGF monoclonal antibody, as previously described (2), or exposed to genistein or to the anti-p75NTR antibody. A representative p53 immunostaining is illustrated in Fig. 6Go. p53 immunoreactivity, which in untreated R cells was distributed in both the nucleus and cytoplasm with high nuclear staining (panel A), underwent cytoplasmic retention both during NGF deprivation (panel B) and genistein treatment (panel C), but not during inactivation of p75NTR (panel D). These data suggest that NGF-dependent p53 nuclear localization in human prolactinomas is mediated by the trkA receptor in a p75NTR-independent way.



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  		Fig. 5. Inactivation of trkA Prevents NGF-Mediated p53 Nuclear Translocation in NR Cells

A, Cells were fixed in methanol at -20 C and processed for p53 immunostaining. Panel a, subcellular localization of p53 in untreated NR cells detected at x40 magnification; panel e, representative NR cells detected at x100 magnification showing that p53 immunostaining is segregated into the cytoplasm; panel b, distribution of p53 immunoreactivity in NGF-treated NR cells detected at x40 magnification; panel f, representative NGF-treated NR cells detected at x100 magnification showing that p53 immunostaining is prevalent into the nucleus; panel c, subcellular distribution of p53 in cells treated with NGF in the presence of the anti- p75NTR antibody (x40 magnification); panel g, x100 magnification of representative NR cells exposed to both NGF and anti-p75NTR antibody showing that p53 immunostaining is more concentrated in the nucleus than in the cytoplasm; panel d, distribution of p53 immunoreactivity in cells treated with NGF in the presence of 1 µg/ml genistein detected at x40 magnification; panel h, x100 magnification of representative NR cells exposed to both NGF and genistein showing that p53 immunostaining is cytoplasmically segregated. Scale bar, 100 µm. B, Effect of genistein on NGF-induced phosphorylation of trkA at Tyr490. A representative WB obtained with the Tyr490-phospho-trkA-specific antibody is shown. Lane 1, NGF-treated cells; lane 2, NGF and genistein-treated cells. C, The efficiency of the anti-p75NTR antibody as an inhibitor of p75NTR-mediated responses was evaluated by EMSA with a specific NF-{kappa}B sequence (9 ). Lane 1, NGF-treated cells; lane 2, NGF- and p75NTR antibody-treated cells. Data are representative of three experiments.

 


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  		Fig. 6. Disruption of NGF-Mediated Autocrine Mechanisms in R Cells Results in p53 Nuclear Exclusion

A, p53 immunoreactivity in representative R cells (x100 magnification) showing either nuclear or both cytoplasmic and nuclear localization. B, p53 immunoreactivity in representative R cells exposed to the anti-NGF antibody (x100 magnification) showing a preferential cytoplasmic localization. C, p53 immunoreactivity in representative R cells exposed to genistein (x100 magnification) showing a preferential cytoplasmic localization. D, p53 immunoreactivity in representative R cells exposed to the anti-p75NTR antibody (x100 magnification) showing both nuclear and cytoplasmic localization. Scale bar, 100 µm.

 
With the aim of identifying the intracellular effectors involved in trkA-mediated p53 nuclear translocation, Erk activation was blocked by PD 98059 (35), and PI3K was inhibited by wortmannin (36). The representative results reported in Fig. 7AGo show that, as in NGF-treated NR cells (panels a and c), also in cells exposed to NGF in the presence of 50 µM PD98059, p53 immunostaining was concentrated into the nucleus (panels b and d), suggesting that this compound did not interfere with NGF action. Control experiments, performed to evaluate the efficiency of PD 98059 treatment, showed that Erk phosphorylation induced by NGF in NR cells was abolished by coincubation with this compound (Fig. 7Go, B and C). Figure 8AGo reports a representative immunocytochemistry demonstrating that 500 nM wortmannin completely blocked p53 nuclear translocation induced by NGF in NR cells. Whereas in NGF-treated NR cells, p53 immunoreactivity was concentrated into the nucleus (panels b and e), in cells exposed to NGF in the presence of wortmannin, as in untreated cells (panels a and d), p53 immunoreactivity was in fact segregated into the cytoplasm (panels c and f). In line with these observations, wortmannin also abolished the stimulatory effect of NGF on p21Cip1/WAF1 expression (Fig. 8Go, B and C). The effect of wortmannin on NGF-induced p21Cip1/WAF1 expression was dose dependent over the range of 5 nM to 0.5 µM Fig. 8DGo). Taken together, these data rule out the possibility that the Ras-Erk pathway may be involved in the control of p53 trafficking elicited by NGF and strongly suggest a crucial involvement of PI3K. To strengthen this finding, we evaluated whether NGF activates PI3K in NR cells by measuring the phosphorylation of its effector Akt (37) in WB experiments with a specific antiphospho(Ser473)-Akt (pAkt) antibody. As reported in Fig. 9Go, exposure of NR cells to NGF resulted in a sustained Akt phosphorylation. Phospho-Akt was hardly detectable in untreated cells (Fig. 9AGo, lane 1) and in cells treated with NGF for 24 h (lane 2). By contrast, pAkt was 6-fold increased after a 2-d NGF treatment (lane 3) and was still high after 5 d (lane 4). Densitometric analysis of three experiments with pAkt normalized to the corresponding values of ß-tubulin is shown in Fig. 9BGo. Because a 5-d administration of the potent PLC{gamma} inhibitor U-73122 was prevented by its toxicity, we investigated whether NGF might activate this enzyme in prolactinoma cells. As reported in Fig. 10Go, a 5-d NGF treatment did not modify the levels of PLC{gamma} measured by WB with a specific antibody (lanes 1 and 2). To evaluate the effect of NGF on PLC{gamma} phosphorylation, protein extracts obtained from untreated or NGF-treated NR cells were immunoprecipitated with an antiphosphotyrosine (p-Tyr) antibody, and the resulting proteins were immunoblotted with a monoclonal anti-PLC{gamma} antibody. Constitutively phosphorylated PLC{gamma} was detectable in NR cells maintained in culture in the presence of serum (lanes 3 and 5). Exposure of these cells to 100 ng/ml NGF for either 5 min or 5 d did not change the levels of phosphorylated PLC{gamma} (lanes 4 and 6). Similar results were obtained in serum-starved cells treated with NGF for 5 min. No specific signals were detectable in either untreated or NGF-treated cells (lanes 7 and 8). Thus PLC{gamma} apparently does not contribute to the effects of NGF on prolactinoma cells.



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  		Fig. 7. The Erk Inhibitor PD 98059 Does Not Affect NGF-Induced p53 Nuclear Translocation in NR Cells

A, p53 immunostaining in NR cells treated with NGF in the absence (panel a) and in the presence (panel b) of 50 µM PD 98059; panels c and d, x100 magnification of representative cells exposed to either NGF (c) or NGF and PD 98059 (d) showing that in both conditions p53 immunoreactivity was preferentially localized into the nucleus. Scale bar, 100 µm. B, 50 µM PD 98059 prevented NGF-induced Erk1/2 phosphorylation in NR cells. Cells were treated with NGF in the absence or in the presence of PD 98059 for different times. Erk1/2 phosphorylation was evaluated by WB with a specific antibody that selectively recognizes the phosphorylated forms of Erk1/2. C, Densitometric analysis of pErk1/2 normalized to the corresponding ß-tubulin levels. Bars represent the mean ± SE of three experiments. *, P < 0.001 vs. NGF-treated cells, Student’s t test.

 


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  		Fig. 8. Inhibition of PI3K Abolishes NGF-Induced p53 Nuclear Translocation and p21Cip1/WAF1 Expression in NR Cells

Cells were treated with 100 ng/ml NGF for 5 d in the absence or in the presence of the PI3K inhibitor wortmannin (0.5 µM), and p53 cellular localization was evaluated by immunocytochemistry (A). In untreated cells p53 immunoreactivity was segregated into the cytoplasm (panels a and d), whereas in NGF-treated cells it was preferentially localized into the nucleus (panels b and e). In cells exposed to both NGF and wortmannin p53 was sequestered into the cytoplasm (panels c and f). Scale bar, 100 µm. B, Representative WB showing the expression of p21Cip1/WAF1 in untreated cells and in cells exposed to NGF in the absence or in the presence of wortmannin. Lane 1, Untreated cells; lane 2, NGF-treated cells; lane 3, NGF- and wortmannin-treated cells. C, Densitometric analysis of p21Cip1/WAF1 normalized to the corresponding ß-tubulin levels. Bars represent the mean ± SE of three experiments. *, P < 0.001 vs. untreated cells, Student’s t test. D, Dose-response curve of wortmannin effect on NGF-induced p21Cip1/WAF1 expression.

 


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  		Fig. 9. NGF Promotes Akt Phosphorylation in NR Cells

Cells were treated with NGF for different times, and Akt phosphorylation was evaluated by WB with a specific antiphospho-Akt (Ser473). A, Representative WB showing the levels of pAkt. Lane 1, Untreated cells; lane 2, 24-h NGF treatment; lane 3, 2-d NGF treatment; lane 4, 5-d NGF treatment. B, Densitometric analysis of pAkt normalized to the corresponding ß-tubulin levels. Bars represent the means ± SE of three experiments. *, P < 0.001 vs. untreated cells.

 


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  		Fig. 10. NGF Does Not Activate PLC{gamma} in NR Prolactinoma Cells

To evaluate PLC{gamma} expression, cytoplasmic proteins were analyzed by WB. To evaluate PLC{gamma} phosphorylation, aliquots of cytoplasmic proteins were immunoprecipitated (IP) with the antiphosphotyrosine antibody, and the resulting immunocomplexes were detected by WB with the anti-PLC{gamma} antibody. Lanes 1, 3, and 5, Untreated NR cells; lanes 2 and 4, 5-d NGF treatment; lane 6, 5-min NGF treatment; lane 7, serum-starved untreated NR cells; lane 8, NGF treatment of serum-starved NR cells. Data are representative of three experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Disruption of p53 function is frequently correlated with tumorigenesis. Mutations occurring within the DNA-binding domain are the prevalent mechanism of p53 inactivation in half of all human tumors (19). Other mechanisms, however, such as interaction with cellular proteins enhancing p53 degradation (38, 39) or with cytoplasmic anchor proteins that prevent p53 nuclear localization (26, 40, 41, 42, 43), can also nullify p53 activity. Although sequence analysis showed that p53 is wild type in the overwhelming majority of pituitary adenomas (27, 28, 29), accumulation of this protein has been detected in a proportion of these tumors (44, 45, 46), especially the invasive type. The existence of functional defects in p53-dependent pathways in pituitary adenomas, however, has never been clearly demonstrated. In this study we report, for the first time, that the phenotype of human prolactinoma cell lines is related to p53 conformation, cellular localization, and transcriptional activity and that NGF is crucial to maintain p53 in its active form.

A critical feature for tumor suppression is a "wild-type" conformation of p53. Mutations in the p53 DNA-binding domain frequently result in the expression of a protein with an inactive mutant conformation (19). Yet wild-type p53 is characterized by intrinsic flexibility that causes it to transiently adopt either wild-type or mutant conformations in a cell cycle-dependent manner (32, 47). In particular, the wild-type conformation correlates with growth suppression, whereas the mutant tertiary structure promotes proliferation in cells expressing wild-type p53 (47). In this study we report that p53 adopts different conformations in R and NR prolactinoma cell lines, due to the presence or the absence of secreted NGF in the culture media. In particular, in the more differentiated, slowly proliferating, NGF-secreting R cells, p53 adopts the wild-type conformation, whereas in the fast proliferating NR cells, which do not produce NGF, p53 takes on the mutant conformation. Moreover, according to previous data obtained in fibroblast and neuroblastoma cell lines (21), in prolactinoma cells a close correlation was also found between p53 conformation and its cellular localization. The wild-type p53 form was present, in fact, in both the nucleus and the cytoplasm in R cells, whereas the mutant p53 form was cytoplasmically segregated in NR. Thus, in the slowly proliferating R cells p53 exhibits functional conformation and localization, but in the fast-proliferating NR cell line, p53 is inactivated by conformational mutation and cytoplasmic segregation. Along this line, aberrant cytoplasmic sequestration of p53 has been detected in many human tumors (22, 48, 49, 50, 51). Interestingly, p53 structural alteration in prolactinomas is reversible and depends on NGF. Inactivation of secreted NGF resulted, in fact, in p53 nuclear exclusion in R cells, whereas NGF administration to NR cells refolded p53 into the wild-type conformation, promoted its nuclear translocation, and restored its specific DNA-binding activity as demonstrated by the transcriptional activation of p21Cip1/WAF1 and Mdm2. Moreover, as a result of p21Cip1/WAF1 expression, cyclin A, cyclin E, and cdk2 were remarkably down-regulated in NGF-treated cells, an effect related to cell cycle arrest at the G1 to S transition (18) that may represent the mechanism of the reported antiproliferative effect of NGF on these cell lines (1). Along this line, NGF-induced inhibition of cell proliferation in PC12 cells (12) and in NIH-3T3 cells (13) has been reported to be mediated by activation of p21Cip1/WAF1. Taken together these data suggest that escape of NR cells from NGF control results in a reversible stabilization of p53 in the mutant conformation likely leading to their fast-proliferating phenotype. It is worth noting that the effects of NGF on p53 conformation, cellular localization, and transcriptional activity were detectable after a long-term treatment. Although this could be related to the doubling time of NR cells (~2 d), the possibility cannot be excluded that other factors induced by NGF, rather than NGF itself, might regulate p53 conformation.

NGF transduces its effects by interacting with two types of surface receptors. From a functional point of view, signaling by trkA and p75NTR, which are often present on the same cell, may be synergistic, independent, or antagonistic. We have previously shown that the p75NTR receptor plays a critical, trkA-independent role in the control of D2 receptor expression in prolactinoma cell lines (9). In this paper we demonstrate that trkA is the receptor that, in a p75NTR-independent way, mediates the effect of NGF on p53 conformation and nuclear localization. Taken together, these data thus suggest that in prolactinoma cell lines, even if trkA and p75NTR are coexpressed (1, 2), they mediate individual and independent functions of NGF. Whether p75NTR and trkA interact to mediate other effects of NGF on these cells remains to be established. The role of trkA in the control of tumor growth is still controversial. TrkA operates, in fact, as a typical mitogenic growth factor receptor in some tumor cell lines (10, 11) but induces differentiation and inhibition of cell growth in others (12, 13, 14). These observations, together with our present data, suggest that the functional effects of trkA are related to the cellular background in which it is expressed, rather than to its intrinsic signaling properties.

NGF binding to trkA results in the activation of different signaling effectors. Our analysis with specific inhibitors and by measuring Akt phosphorylation strongly pointed to PI3K as the most plausible intracellular messenger mediating the effects of NGF on the p53/p21Cip1/WAF1 pathway. PI3K has been implicated in disparate cell responses including protection from apoptosis, stimulation of cell proliferation, and tumor formation (37). Nevertheless, there is evidence that in different tumor cell lines PI3K is required for p53-dependent induction of p21Cip1/WAF1 in response to antitumor agents (53, 54, 55). Our data, showing that in prolactinoma cell lines the trkA-PI3K pathway is involved in the activation of p21Cip1/WAF1 in response to NGF, are in line with these observations.

In conclusion, our data, showing that the tertiary structure and cellular localization of wild-type p53 are related to the degree of transformation of human prolactinoma cell lines, suggest that disruption of p53 function by mechanisms different from genomic mutation might contribute to the development of these tumors. Moreover, the finding that p53 conformation may be shifted from mutant-like to wild type by NGF suggests that this structural alteration is reversible, an observation that might open new therapeutic perspectives, if such a defect is found also in primary pituitary adenomas.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Prolactinoma Cell Cultures and Treatments
R and NR prolactinoma cells were grown in Ham’s F10 medium supplemented with 2.5% fetal bovine serum, 15% horse serum (Euroclone, Celbio, Milano, Italy) 4 mM glutamine, and 100 U of penicillin-streptomycin at 37 C and 5% CO2. NR cells were treated with 100 ng/ml NGF (2.5S, mouse, Alomone Labs, Jerusalem, Israel) for 5 d in the absence or in the presence of either mouse anti-p75NTR monoclonal antibody (100 ng/ml; Chemicon International, Temecula, CA), or PD98059 (50 µM; Calbiochem, San Diego, CA) or genistein (1 µg/ml; BIOMOL Research Laboratories, Inc., Plymouth, PA) or wortmannin (0.5 µM, Calbiochem). R cells were exposed either to the anti-NGF antibody (50 ng/ml; Chemicon) or to 1 µg/ml genistein or to the anti-p75NTR antibody (100 ng/ml). NGF and genistein were added once at the beginning of treatment, whereas the anti-p75NTR antibody, the anti-NGF antibody, PD 98059, and wortmannin were added to the cultures every day.

Immunoprecipitation and WB
Cells were lysed in 50 mM Tris-HCl (pH 7.6) containing 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P-40 (NP-40), 1 mM NaF, 1 mM Na3VO4, and a mixture of protease inhibitors (Complete Mini Protease Inhibitors, Roche Molecular Biochemicals, Basel, Switzerland) and centrifuged at 18,000 x g for 20 min at 4 C. The resulting supernatants containing cell proteins were stored at -20 C. Aliquots of proteins (10–30 µg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. After blotting for 1 h at room temperature in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% low-fat dry milk, membranes were incubated overnight at 4 C using the following antibodies: anti-p53 monoclonal antibody PAb421 (1.5 µg/ml) (Oncogene Science, Inc., Boston, MA); anti-p21Cip1/WAF1 monoclonal antibody (1 µg/ml) (PharMingen International, San Diego, CA); anti-phospho-p44/42 Erk (Thr202/Tyr204) polyclonal antibody (1:1,000 dilution) (Cell Signaling, Beverly MA); anti-cdk2 monoclonal antibody (1:200 dilution) (Santa Cruz Biotechnology, Heidelberg, Germany); anti-phospho-trkA (Tyr490) polyclonal antibody (1:1,000 dilution in Tris-buffered saline containing BSA 5% and Tween 20 0.1%) (Cell Signaling); antiphospho-Akt (Ser473) antibody (Cell Signaling); anti-cyclin E monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY). To ensure equal protein loading, membranes were stripped with a WB recycling kit (Chemicon International) and reprobed with anti-ß-tubulin antibody (1:1,500 dilution) (Neo-Markers, Fremont, CA).

To isolate nuclear and cytoplasmic proteins, cells were resuspended in ice-cold 10 mM HEPES (pH 7.9) containing 1.5 mM MgCl2, 10 mM KCl, and the protease inhibitors, incubated on ice for 5 min, added with 10% NP-40, mixed by vortex for 10 sec, and centrifuged at 5,000 x g for 5 min. The supernatants containing the cytoplasmic proteins were stored at -20 C, and the pellets were resuspended in ice-cold 20 mM HEPES (pH 7.9) containing 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and the protease inhibitors, shaken for 15 min at 4 C, and centrifuged at 18,000 x g for 20 min at 4 C. The supernatants containing the nuclear proteins were stored at -20 C. Aliquots of cytoplasmic (15 µg/lane) and nuclear proteins (30 µg/lane) were resolved by SDS-PAGE and processed as described above to detect p53. To ensure equal protein loading, membranes were stripped and reprobed with ß-actin antibody (1:500 dilution) (Sigma, St. Louis, MO) for the nuclear fraction and with ß-tubulin antibody (1:1,500 dilution) for the cytoplasmic fraction. For detection, an enhanced chemiluminescence system (Amersham International, Cardiff, UK) was used with horseradish peroxidase (HRP)-conjugated secondary antibodies (antirabbit, 1:2,000 dilution, Santa Cruz Biotechnology, Inc.; and antimouse, 1:1,500 dilution, DAKO, Copenhagen, Denmark). To detect Tyr490-phosphorylated trkA, an enhanced chemiluminescent system allowing visualization of proteins in the low femtogram range (Supersignal West, Pierce Chemical, Milano, Italy) with a HRP-conjugated secondary antibody (Pierce Chemical) was used. The blots obtained from at least three independent experiments were analyzed by densitometry, and the specific signals were normalized to the corresponding ß-tubulin or ß-actin staining.

To detect tyrosine-phosphorylated PLC{gamma}, aliquots of the supernatant containing the cytoplasmic proteins (100 µg) were immunoreacted overnight at 4 C with 2 µg of a monoclonal antiphosphotyrosine (pTyr) antibody (Transduction Laboratories, Inc., Lexington, KY). The resulting immunocomplexes were precipitated by incubation with protein A-Sepharose (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The pellets were resuspended in 50 mM Tris-HCl, pH 7.6, containing 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 1 mM NaF, 1 mM Na3VO4, and the protease inhibitors, centrifuged at 18,000 x g at 4 C, resuspended in 20 µl of sample buffer and resolved by SDS-PAGE. Membranes were incubated overnight at 4 C with the monoclonal anti-PLC{gamma} antibody (1:250 dilution, Transduction Laboratories, Inc.). The immunoreaction was detected by enhanced chemiluminescence as previously described.

Conformation-Specific Immunoprecipitation of p53
Conformation-specific immunoprecipitation of p53 was performed according to Verhaegh (33). Cells were lysed in the immunoprecipitation nondenaturing buffer (10 mM Tris, pH 7.6, containing 140 mM NaCl and 0.5% NP-40 and protease inhibitors) for 20 min on ice and cleared from cell debris by centrifugation. To prevent nonspecific binding, the supernatant was precleared with 10% protein A-Sepharose (Santa Cruz Biotechnology, Inc.) for 20 min on ice, followed by centrifugation. For immunoprecipitation of p53, 1 µg of the monoclonal antibodies PAb1620 (wild type specific, Oncogene Science, Inc.), PAb240 (mutant specific, Neo-Markers), or PAb1801 (recognizing both wild-type and mutant p53, Oncogene Science, Inc.) was added to the precleared supernatant for 2 h at 4 C. Immune complexes were collected with protein A-Sepharose (Santa Cruz Biotechnology, Inc.) and washed five times with immunoprecipitation buffer. Immunoprecipitated p53 was detected by WB using the anti-p53 polyclonal antibody CM-1 (1:1200 dilution) (Novocastra, Newcastle, UK), which recognizes both mutant and wild-type p53 and a HRP-conjugated secondary antibody. Data obtained from three independent experiments were analyzed by densitometry and expressed as the ratio between PAb1620- and PAb240-immunoprecipitated p53.

Immunocytochemistry
Cells were plated at low density on poly-L-lysine-coated glass cover slips, fixed with methanol at -20 C for 5 min, and permeabilized for 20 min at room temperature in PBS containing 0.2% Triton X-100 and 10% normal rabbit serum (DAKO Corp.). Cells were incubated overnight at 4 C with the monoclonal anti-p53 antibody PAb421 (20 µg/ml in PBS containing 0.2% Triton X-100 and 1% normal rabbit serum) and then with the biotinylated rabbit antimouse secondary antibody (1:400 dilution in PBS containing 0.2% Triton X-100 and 1% normal rabbit serum) (DAKO Corp.) for 1 h at room temperature. After three rinses with PBS, cells were incubated with the avidin-biotin complex (ABC, DAKO Corp.) for 45 min at room temperature. Peroxidase staining was obtained by incubation in 0.06% 3,3-diaminobenzidine and 0.01% H2O2 in PBS buffer. Cells were visualized at both x40 and x100 magnification.

RNA Isolation and RT-PCR
Total RNA was isolated using the Nucleospin-RNA II Kit (CLONTECH, Palo Alto, CA). Two micrograms of each sample were transcribed into cDNA by using the murine Moloney leukemia virus reverse transcriptase (Promega Corp., Madison, WI) and oligo(dT)18 as a primer. p53 was amplified with the oligonucleotides 5'-CTGAGGTTGGCTCTGACTGTACCACCATCC-3' and 5'-CTCATTCAGCTCTCGGA ACATCTCGAAGCG-3', which generate a 371-bp fragment. The reaction was performed for 28 cycles (94 C, 1 min; 66 C, 30 sec; 72 C, 1 min). Mdm2 was amplified for 28 cycles (94 C, 1 min; 52 C, 30 sec; 72 C, 1 min) with the oligonucleotides 5'-GAAAGAGGTTCTTTTTTATCTTGG-3' and 5'-ATTTTCTTCTGTCTCACTAATTGC-3', which generate a 364-bp fragment. To amplify cyclin A, the oligonucleotides 5'-TCCTTGGAAAGCAAACAGTAAA-3' and 5'-AACCCACTTTAGGTTTACATTT-3', generating a 332-bp fragment, were used, and the reaction was performed for 35 cycles (94 C, 1 min; 50 C, 30 sec; 72 C, 4 min). Amplification with 5'-TAAAGACCTCTATGCCAACACAGT-3' and 5'-CACGATGGAGGGCCGGACTCATC-3' primers encoding a fragment of human ß-actin (94 C, 1 min; 60 C, 30 sec; 72 C, 1 min; 25 cycles) was performed as a control of the amount of cDNA in each sample. All reactions were performed within the linear range of amplification. Omission of the reverse transcription was performed as a control of the PCR specificity. The PCR products were analyzed on 1% agarose gels stained with ethidium bromide.


   FOOTNOTES
 
This work was supported by grants from Ministero Università e Ricerca (MIUR 9906153187 and MIUR 2002067251) and by Fondazione Italiana per la Ricerca sul Cancro (FIRC) to C.M.

Abbreviations: cdk, Cyclin-dependent kinase; HRP, horseradish peroxidase; NF-{kappa}B, nuclear factor {kappa}B; NGF, nerve growth factor; NP-40, Nonidet P-40; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipases C; WB, Western blot.

Received for publication May 23, 2003. Accepted for publication September 26, 2003.


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M. P. Gillam, M. E. Molitch, G. Lombardi, and A. Colao
Advances in the Treatment of Prolactinomas
Endocr. Rev., August 1, 2006; 27(5): 485 - 534.
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