Introduction

The nm23 gene family includes two murine and six human genes1 (and references therein).2 The first, referred to as nm23-M1, was isolated on the basis of its reduced expression in highly metastatic murine melanoma cell lines, as compared with their nonmetastatic counterparts.3 Afterward, a crucial role in blocking metastasis progression has been suggested for the murine nm23-M1 and the human nm23-H1 genes that share 88% nucleotide identity and encode proteins 95% identical1,4 (and references therein). Nevertheless, in tumours such as childhood neuroblastoma, a high expression of nm23 positively correlates with tumor aggressiveness.5,6 Moreover, a serine 120 to glycine mutation in the Nm23-H1 sequence, that impairs proper protein folding,7 occurs in aggressive childhood neuroblastoma.8 Noteworthy, the mutant nm23-H1S120G cDNA transduced into human breast carcinoma cells, fails to inhibit cell motility,9 a fundamental component of the metastatic phenotype. The inhibition of cell motility is also abrogated by the transduction of the mutant nm23-H1P96S cDNA.9 This mutant reproduces the killer of prune (k-pn) mutation of the awd gene, the nm23 homologue in Drosophila melanogaster.10 The awd k−pn mutation is lethal in the genetic context of the null mutation of the prune (pn) gene involved in the eye pigment synthesis.11

The nm23 genes encode nucleoside diphosphate kinases (NDPK), exameric enzymes that catalyse the exchange of γ-phosphates between tri- and diphosphonucleosides.12 The reaction involves the transient phosphorylation of a conserved histidine, namely histidine 118 in the human enzymes.13 The NDPK activity does not correlate with the suppression of the metastatic phenotype.14

Several observations suggest an involvement of the nm23 genes in development and differentiation1 (and references therein). Specifically, their action is mainly relevant to the functional differentiation of epithelia and neural tissues.15,16,17 We have demonstrated previously that the overexpression of nm23-M1 in rat pheochromocytoma PC12 cells enhances susceptibility to nerve growth factor (NGF)-induced sympathetic neuronal cell differentiation by inhibiting proliferation and stimulating neurite outgrowth.18 Upon NGF treatment, stable nm23-M1 PC12 transfectants undergo growth arrest, express cytoskeletal proteins specific to neuritogenesis and display branching neurites within 4 days, whereas parental PC12, as well as control transfectants, require at least 8 days. Stable antisense nm23-M1 PC12 transfectants, instead, show a marked increase in the proliferative rate and are inhibited in undergoing differentiation.

Growth arrest being a prerequisite for cell differentiation, genes that negatively regulate the transition from the G1 to the S-phase of the cell cycle are involved in such a phenomenon. A crucial role is played by those belonging to the family of the retinoblastoma (RB) gene, the prototype of the oncosuppressors19,20 (and references therein). Their products, pRB, p107 and pRb2/p130, are phosphoproteins that control the function of transcription factors such as those of the E2F family mainly responsible for the S-phase entry21 (and references therein). Moreover, the RB family genes regulate embryonic development and differentiation22 (and references therein). As far as Rb2/p130 is concerned, it has been suggested that it might play a major role in maintaining the differentiated state, due to the ability of the gene product to form (with the E2F family members) stable complexes at late stages of the differentiation process.23,24,25,26,27 Nevertheless, Rb2/p130 overexpression is able to induce differentiation of neuroblastoma cells.28 High expression levels of pRb2/p130 are detectable in terminally differentiated skeletal muscle and nervous tissue.29 In PC12 cells, NGF treatment increases pRb2/p130 endogenous expression levels and Rb2/p130 overexpression is able per se to trigger the onset of differentiation.30

In this scenario, in order to elucidate the mechanisms by which the modulation of the antimetastatic nm23 gene influences PC12 cells proliferation and differentiation, we approached, in such model system, the analysis of the negative regulators of the cell cycle G1/S transition. The results herein shown provide the first evidence of interplay between nm23 and Rb2/p130 in committing PC12 cells to a more sensitive state for neuronal differentiation.

Results

Expression of the RB family proteins in stable sense and antisense nm23-M1 PC12 transfectants

PC12 cells exposed to NGF stop dividing within 7–8 days and gradually develop the phenotype of sympatethic neurons.31,32 Stable nm23-M1 PC12 transfectants, in the presence of NGF, rapidly accumulate in the G0/G1 phase of the cell cycle undergoing early morphological differentiation after 4 days of treatment. On the contrary, stable antisense nm23-M1 PC12 transfectants, upon NGF treatment, continue to proliferate without differentiating.18 In this model system we investigated the expression and postranslational modifications of the RB proteins, because of their role as negative regulators of the cell cycle G1/S transition. Protein analysis was performed on cell lysates from different control, sense and antisense stable nm23-M1 PC12 transfectants,18 both cycling and NGF-treated. Figure 1 shows Western blots chosen as representative of the experiments performed with the different clones. In cycling control and sense transfectants the bands corresponding to pRb, pRb/p130 and p107 displayed, as expected, a microheterogeneous pattern due to the presence of species with different degrees of phosphorylation. In cycling antisense transfectants the bands corresponding to the RB proteins displayed a majority of hyperphosphorylated slow-migrating forms. After 4 and 8 days of NGF-treatment, pRb and p107 showed a similar increase in the hypophosphorylated fast-migrating forms in both control and sense transfectants. The pattern of pRb2/p130 did not change between 4- and 8-day NGF-treated control transfectants, instead a dramatic increase in the protein level, in its underphosphorylated form, was detected in NGF-treated sense transfectants. The increase in pRb2/p130 expression was more evident after 4 days of NGF treatment coincident with the acquisition of the differentiated phenotype by the stable sense nm23-M1 PC12 transfectants.18 NGF-treated antisense transfectants showed a microheterogeneous pattern of all the RB proteins in agreement with their continuous proliferative activity.18

Figure 1
figure 1

Western blot analysis of the RB pocket proteins in cycling and NGF-treated control, sense (S-nm23-M1) and antisense (AS-nm23-M1) PC12 transfectants. Aliquots of cell lysates containing 30 μg of proteins were resolved by 6% SDS–PAGE. The analysis shown is representative of at least three experiments per each detection, performed with different clones

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Regulation of the Rb2/p130 promoter activity by nm23-M1 in PC12 cells

The marked increase in pRb/p130 expression observed in stable nm23-M1 PC12 transfectants upon NGF treatment, prompted us to investigate whether it was due to an increase in the transcription of the Rb2/p130 gene. For this purpose, PC12 cells were cotransfected with the plasmid containing the minimal promoter region of the Rb2/p130 gene, encompassing 201 nucleotides prior to the translation codon, upstream the CAT reporter gene (hereinafter referred to as −201 CAT construct),30 together with either the empty control vector (pcDNA3), or the plasmid encoding nm23-M1 (pcDNA3-S-nm23-M1),18 or the plasmid containing the nm23-M1 coding region in the antisense orientation pcDNA3-AS-nm23-M1.18

Figure 2 shows the histogram corresponding to the CAT activity detected 2 days after the transfection with each couple of vectors in growing condition (Figure 2A) and upon NGF treatment (Figure 2B). The cotransfection of nm23-M1 increased the CAT activity by about 35 and 50% in cycling and in NGF-treated cells, respectively as compared to the basal CAT activity. On the contrary, the cotransfection of antisense nm23-M1 inhibited the basal CAT activity by about 45 and 20%, respectively. These data indicate that in PC12 cells Rb2/p130 transcription can be modulated by different nm23 levels.

Figure 2
figure 2

Modulation of RB2/p130 promoter activity by nm23-M1 in PC12 cells. PC12 cells were cotransfected with 6 μg of the −201 CAT construct plus 12 μg of pCDNA3 (control), or pCDNA3-S-nm23-M1, or pCDNA3-AS-nm23-M1 plus 3 μg of pCMV-β-gal. Following transfection, cells were maintained in growth medium (A) or NGF-treated (B) for 48 h. Afterward, cell extracts, normalised for β-galactosidase activity, were assayed for CAT activity. Histograms in (A) and (B) show the CAT activities detected for each condition relative to the control activity that was arbitrarily assigned value 100. Data represent the average of five experiments, each in duplicate, for each condition

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Regulation of Rb2/p130 promoter activity and endogenous pRb2/p130 levels by nm23-H1 and nm23-H1 mutants in PC12 cells

In a first attempt to identify in the nm23 sequence the residues required for the induction of Rb2/p130 transcription, we also investigated the effect of different mutants of the human nm23-H1 gene on the Rb2/p130 promoter as compared with wild-type nm23-H1. PC12 cells were cotransfected with the −201 CAT construct together with either the empty control vector (pCMV) or the plasmid containing the nm23-H1 coding region (pCMV-nm23-H1)9 or one of the following plasmids containing mutant nm23-H1 cDNAs: pCMV-nm23-H1H118F (hereinafter referred to as pCMV-H118F), pCMV-nm23-M1S120G (hereinafter referred to as pCMV-S120G), pCMV-nm23-H1P96S (hereinafter referred to as pCMV-P96S).9 Histidine 118 is involved in Nm23 NDPK activity;13 serine 120 is the site of the mutation found in human neuroblastoma;8 and proline 96 corresponds to the site of the k-pn mutation of the Drosophila awd gene.11

Figure 3 shows the histograms corresponding to the CAT activity detected two days after the transfection with each couple of vectors in cycling condition (Figure 3A) or upon NGF treatment (Figure 3B). In cycling cells the cotransfection of nm23-H1 increased the CAT activity of 70% above the basal level, whereas the H118F mutant had no effect. The S120G and the P96S mutants inhibited the basal CAT activity of about 25 and 35%, respectively (Figure 3A). In NGF-treated cells the cotransfection of nm23-H1 increased the CAT activity of 25% above the control level. The H118F mutant, that was ineffective in cycling cells, increased the CAT activity of about 20%. The S120G and the P96S mutants, instead, inhibited the CAT activity of 20 and 10%, respectively (Figure 3B). We also investigated the effects of nm23-H1 and its mutants on Rb2/p130 translation in PC12 cells. Lysates from PC12 cells transiently transfected with either nm23-H1 or each of the different mutants were normalised on the basis of an equivalent expression of the endogenous and exogenous Nm23 proteins, as evaluated by Western blotting (not shown), and then analyzed for the expression of endogenous pRb2/p130. Figure 4 shows that the levels of the Rb2/p130 gene product detected in cycling and in NGF-treated PC12 transfectants 2 days after the transfection, were strongly inhibited by the S120G and P96S mutants, as also indicated by the reported relative density values.

Figure 3
figure 3

Modulation of RB2/p130 promoter activity by nm23-H1 and nm23-H1 mutants in PC12 cells. PC12 cells were cotransfected with 6 μg of the −201 CAT construct plus 12 μg of pCMV (control), or pCMV-nm23-H1, or pCMV-H118F, or pCMV-S120G, or pCMV-P96S plus 3 μg of pCMV-β-gal. Following transfection, cells were maintained in growth medium (A) or NGF-treated (B) for 48 h. Afterward, cell extracts, normalised for β-galactosidase activity, were assayed for CAT activity. Histograms in (A) and (B) show the CAT activities detected for each condition relative to the control activity that was arbitrarily assigned value 100. Data represent the average of five experiments, each in duplicate, for each condition

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Figure 4
figure 4

Modulation of Rb2/p130 translation by nm23-H1 and nm23-H1 mutants in PC12 cells. Western blot analysis of pRb2/p130 in transient PC12 transfectants. PC12 cells were transfected with 25 μg of pCMV (control), or pCMV-nm23-H1, or pCMV-H118F or pCMV-S120G, or pCMV-P96S. Following transfection, cells were maintained in growth medium (left) or NGF-treated for 48 h (right). Afterward, aliquots of cell lysates, normalised for endogenous and exogenous Nm23 protein expression, were resolved by 6% SDS–PAGE. Relative density values (rdv) of the detected bands are reported. The analysis shown is representative of at least three experiments, performed with different transfectants

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Inhibition of PC12 cells differentiation by nm23-H1 S120G and P96S mutants

We also verified the effect on NGF-induced PC12 differentiation elicited by the expression of the nm23-H1 mutants, as compared with wild-type nm23-H1. PC12 cells were cotransfected with the plasmids containing nm23-H1 or the mutants in combination with a β-galactosidase reporter construct, as a transfection marker, and cultured in NGF containing medium. Four days after transfection, cells were stained for the β-galactosidase activity and a minimum of 200 positive cells were observed per each experiment. Cells were scored as differentiated when neuritic length exceeded at least twofold the cell body. Figure 5 shows the morphology of the PC12 cells transfected with the control pCMV vector, the pCMV-nm23-H1, the pCMV-H118F, the pCMV-S120G, and the pCMV-P96S plasmids upon 4-day NGF treatment. The histogram represents the percentage of differentiated cells observed for each type of transfection. It appears evident that nm23-H1, as its murine cognate nm23-M1,18 enhanced neuronal differentiation of PC12 cells and that the NDPK-defective H118F mutant was similarly able to promote neuritogenesis. On the contrary, the S120G and P96S mutants inhibited NGF induced differentiation.

Figure 5
figure 5

Effects of nm23-H1 and nm23-H1 mutants on NGF-induced neurite outgrowth in PC12 cells. PC12 cells were cotransfected with 20 μg of pCMV (control), or pCMV-nm23-H1, or pCMV-H118F, or pCMV-S120G, or pCMV-P96S plus 4 μg of pCMV-β-gal. After 4 day-NGF treatment, cells were stained for β-galactosidase activity. Two hundred positive cells per transfection were scored as differentiated when neuritic length exceeded at least twofold the cell body. Arrows indicate β-galactosidase positive cells. Data in the histograms represent the mean of three experiments, each in duplicate, for each condition

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Discussion

We have demonstrated previously that the murine antimetastatic nm23 gene is able to promote NGF-induced differentiation of PC12 cells.18 Cell cycle withdrawal is mandatory for differentiation in many cell types. Accordingly, NGF-treated PC12 cells overexpressing nm23 rapidly accumulate into the G0/G1 phase.18 The present study was aimed at establishing the link between the nm23 gene and the regulatory machinery of cell cycle during PC12 neuronal differentiation.

The genes of the retinoblastoma family, namely RB, p107 and Rb2/p130 are negative regulators of the transition between G1- and S-phases of the cell cycle and are crucial effectors in embryo development and cell differentiation. Their products are phosphoproteins that in the hypophosphorylated form modulate the function of several nuclear transcription factors mainly of the E2F family.

We investigated the expression of the members of the RB family in PC12 clones stably transduced with either sense or antisense nm23-M1 cDNA.18 Upon NGF treatment, as expected, a general increase in the dephosphorylation of the RB proteins was observed in control and sense nm23-M1 PC12 transfectants. In contrast, in the antisense nm23-M1 transfectants the three RB proteins displayed a pattern of hyperphosphorylated forms. The hyperphosphorylated forms being inactive as negative regulators of the cell cycle, this observation reflected the high proliferative activity shown by the antisense nm23-M1 PC12 transfectants despite the NGF treatment.18 Moreover, beside the increased dephosphorylation, a striking accumulation of pRb/p130 was also evident in the sense nm23-M1 PC12 transfectants.

Since NGF treatment of PC12 cells upregulates the endogenous Nm2318 and pRb2/p13030 proteins, and Rb2/p130 overexpression activates the PC12 differentiation program30, we investigated whether the modulation of the expression of nm23-M1 in PC12 cells could result into a modulation of the Rb2/p130 promoter. Our results indicated that in the PC12 model, the activity of the Rb2/p130 promoter was modulated with relation to the expression levels of nm23-M1. The stimulation of Rb2/p130 transcription, occurring upon NGF treatment of PC12 cells, was clearly enhanced by the transfection of nm23-M1 and, on the contrary, inhibited by the transfection of the antisense nm23-M1 cDNA. Moreover, the human nm23-H1 was effective in stimulating the activity of the promoter of Rb2/p130 and the synthesis of the gene product as well.

The synthesis of nucleoside triphosphates does not appear to support the role of nm23 in the suppression of the metastatic phenotype.14 We verified the effect of the NDPK defective H118F mutant on Rb2/p130 transcription and on PC12 differentiation. The mutant was still effective in increasing the Rb2/p130 promoter activity upon NGF treatment, even if at a lesser extent than wild-type nm23-H1. Accordingly, the H118F mutant was as able as wild-type nm23-H1 to up-regulate the endogenous expression of pRb2/p130 and to promote NGF-induced differentiation in PC12 cells. These results indicate that the regulation of the NGF-induced differentiation mediated by nm23 does not require the NDPK activity of the nm23 gene product, thus resembling what observed as far as cell motility inhibition,14 a parameter that well correlates with the metastatic potential of tumour cells. These data are in contrast with those described by other authors that reported a suppression of NGF- and dibutyryl cyclic AMP-induced differentiation of a PC12 sub-cell line.33 They showed a slight decrease of neurite outgrowth upon transient transfection of a NDPK defective mutant and a marked inhibition of differentiation in stably transfected clones, as compared to wild-type NDPK transfection. The discrepancy between the results might be partly due to a clonal peculiarity of the PC12 sub-cell line and to a shorter treatment period prior to evaluating the morphological differentiation. An inhibitory effect on the Rb2/p130 promoter activity was instead consequent to the expression of the S120G and P96S mutants. In agreement, the expression of PC12 endogenous pRb2/p130 was downregulated. Furthermore, a striking inhibition of NGF-induced PC12 differentiation was observed as comparing the morphology, upon NGF treatment, of S120G and P96S transfectants with control PC12 transfectants.

The serine 120 to glycine mutant NDPK retains the enzymatic activity, instead protein stability to denaturation is greatly compromised34 leading to an incorrect protein folding.7 Moreover, the mutation alters subunits interaction, resulting in the preferential formation of dimers rather than examers, relatively to the wild-type, which may affect interaction with other cellular factors.34 The proline 96 to serine mutation mimics the k-pn mutation of awd, the Drosophila nm23 homologue. The k-pn mutation alters protein assembly35 and the structure of the so-called k-pn loop that is important for NDPK subunits interaction and plays an important role in the formation and stability of the examers.35,36 Noteworthy, our data indicated serine 120 and proline 96 to be of crucial importance for Nm23-H1 to promote PC12 differentiation and to up-regulate Rb2/p130 expression. Since both mutations affect subunits interaction, it can be postulated that the regulatory function exerted by the Nm23-H1 protein in cell differentiation strictly requires the formation of high affinity examers that allow the interaction with other factors. In view of this and considering that Nm23-H1 has not been demonstrated as a transcription factor, it can be supposed that the effect on Rb2/p130 promoter activity, observed upon nm23-H1 transfection of PC12 cells, is mediated by the interaction of the Nm23 protein with regulators of Rb2/p130 transcription. It is conceivable that the expression of the S120G and P96S nm23-H1 mutant cDNAs impairs proper protein complexes assembly resulting in the inhibition of the differentiative process and of Rb2/p130 expression. Moreover, taking into account the effects on cell motility observed upon S120G or P96S cDNA transfection,9 our results suggest that nm23 antimetastatic and differentiative functions share a common pathway.

In synthesis, the present study, aimed to verify a putative interplay between nm23 and the negative regulators of the cell cycle, demonstrated the Rb2/p130 gene as a partner in driving neuronal differentiation of PC12 cells. The importance of Rb2/p130 in PC12 cells differentiation as an effector downstream the AP-2 differentiation program, has been demonstrated.30 AP-2 is a transcription factor implicated in embryogenesis37,38 and in the ectodermal differentiation program.39,40 It can be argued that nm23 enhances the susceptibility of PC12 cells to NGF-induced differentiation by promoting the onset of the differentiation program that involves AP-2 and Rb2/p130. Of note, we have herein shown that the transfection of the AS-nm23-M1 cDNA into PC12 cells, besides having an inhibitory effect on NGF-induced differentiation,18 also downregulates Rb2/p130 promoter activity. In this context, in order to better define the specific role of nm23 in driving the neuronal differentiation program, it will be of relevance to investigate whether the antimetastatic gene operates in a synergism with AP-2 in positively regulating Rb2/p130 transcription and translation.

Materials and Methods

Cell lines

PC12 cells were the original described by Greene and Tischler.32,33 Stable control, sense and antisense nm23-M1 PC12 transfectants were as described.18 Cell lines were grown in RPMI 1640 (Bio Whittaker) supplemented with 10% heat-inactivated horse serum (Hyclone Laboratories) and 5% foetal bovine serum (Hyclone Laboratories). NGF treatment was performed as previously described18 in RPMI 1640 supplemented with 1% horse serum and 100 ng/ml NGF.

Western blot analysis

Cell lysis and SDS–PAGE were performed as previously described.41 To avoid quantitative errors due to protein degradation, equal quality of cell lysates was verified by staining duplicate gels with Coomassie brilliant blue G-250 (Bio-Rad).18 After protein electroblotting to nitrocellulose,41 complete transfer was verified by staining nitrocellulose with Ponceau S (Sigma).18 Immunodetection was performed by enhanced chemiluminescence (ECL, Amersham). Primary antibodies were the followings: anti-pRb, anti-pRb2/p130, anti-p107 (all from Santa Cruz Biotechnology, Inc.), anti-GST-Nm23-M141 anti-Nm23-H1 (Transduction Laboratories).

Plasmids

Plasmids were the followings: pcDNA3 vector (Invitrogen Corp.); pcDNA3-S-nm23-M1 and pcDNA3-AS-nm23-M1 plasmids containing the full-length nm23-M1 coding region in sense and antisense orientation, respectively;18 pCMV vector (Invitrogen Corp.); pCMV-nm23-H1 containing the full-length nm23-H1 coding region;9 pCMV-nm23-H1H118F, pCMV-nm23-H1S120G, pCMV-nm23-H1P96S containing the H118F, the S120G and the P96S nm23-H1 mutant cDNAs, respectively;9 −201 CAT construct containing the minimal promoter region of the Rb2/p130 gene cloned in the pSV0t2-CAT vector;30 pCMV-β-gal plasmid expressing the β-galactosidase reporter.30

Transient transfections

PC12 cells (2×106) were transfected by electroporation at 300 V and 500 μF using a Bio-Rad Gene Pulser apparatus connected to a Bio-Rad Pulse Controller unit achieving about 30–35% transfection efficiency. When required, the electroporated PC12 cells were plated on collagen coated plates and NGF-treated as above.

CAT assays

The plasmids were individually cotransfected with the −201 CAT construct and the pCMV-β-gal plasmid into PC12 cells. After 48 h, cell extracts were normalised based on β-galactosidase activity and assayed for CAT activity as already described.42

Differentiation assays

The plasmids were individually cotransfected with the pCMV-β-gal plasmid. The transfected cells were NGF-treated for 4 days, stained for β-galactosidase activity and observed under a phase-contrast microscope.