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Cell Growth & Differentiation Vol. 11, 517-526, October 2000
© 2000 American Association for Cancer Research


Articles

Retinoic Acid Induces Neuronal Differentiation of Embryonal Carcinoma Cells by Reducing Proteasome-dependent Proteolysis of the Cyclin-dependent Inhibitor p271

Gustavo Baldassarre, Angelo Boccia, Paola Bruni, Claudia Sandomenico, Maria Vittoria Barone, Stefano Pepe, Tiziana Angrisano, Barbara Belletti, Maria Letizia Motti, Alfredo Fusco and Giuseppe Viglietto2

Servizio Oncologia Sperimentale E, Istituto Nazionale Tumori, 80131 Naples, Italy [G. B., A. B., P. B., T. A., B. B., M. L. M., G. V.]; Cattedra Oncologia Medica, c/o Dipartimento di Oncologia ed Endocrinologia Molecolare e Clinica [C. S., S. P.] and Dipartimento di Biologia e Patologia Cellulare e Molecolare "L.Califano" [M. V. B.], Facoltà di Medicina e Chirurgia, Università di Napoli "Federico II," 80131 Naples, Italy; and Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia di Catanzaro, Università Magna Graecia, 88100 Catanzaro, Italy [A. F.]

Abstract

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 up-regulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.

Introduction

RA,3 a biologically active metabolite of vitamin A, plays a critical role during normal development and regulates growth and/or differentiation in a variety of tumor cell lines (1) . In particular, strong evidence supports a role of RA in neuronal development (2, 3, 4) . EC cells represent a suitable model to investigate the molecular mechanisms involved in RA signaling. A clonal subline derived from the human EC NTERA-2 cell line (i.e., the NT2/D1 cells) exhibits the properties of multipotent stem cells and differentiates into neurons on treatment with RA (5) . RA treatment of NT2/D1 cells results in growth inhibition and neuronal differentiation, which can be monitored by the expression of markers such as cytoskeletal proteins and secretory or surface markers (i.e., A2B5; Refs. 6 and 7 ). RA-differentiated NT2/D1 cells resemble morphologically primary neuronal cultures from rodents and elaborate axons and dendrites (7 , 8) .

Terminal differentiation of cells requires withdrawal from the cell cycle and induction of a novel program of gene expression, which leads to the elaboration of a specialized phenotype (9 , 10) . The factors that determine whether cells continue to proliferate or arrest growth and differentiate operate during the G1 phase of the cell cycle (11, 12, 13) . Progression of a cell into S phase is dependent on the coordinated activation of a small family of serine/threonine kinases, the CDKs (14) . CDKs play a crucial role in the G1 phase, and the regulation of their function is critical for the commitment to cell differentiation. The activity of CDKs is positively regulated by association with activating subunits, the cyclins (15, 16, 17) , and is negatively regulated by a group of inhibitory proteins called CKIs (18) . Thus far, two classes of CKIs have been identified: (a) the INK4 proteins that specifically inhibit cyclin D-CDK4/CDK6; and (b) the Kip/Cip proteins that inhibit most cyclin-CDK complexes (19) . The Kip/Cip family of CKIs contains p21, p27, and p57 (20, 21, 22) . Recent data in the literature have pointed out that CKIs may be implicated in the differentiation of various cell types (23, 24, 25, 26, 27, 28, 29, 30, 31) . In particular, a central role during neuronal differentiation has been proposed for p27: p27 expression strictly correlates with the differentiation grade of neuronal cells, both in vivo and in vitro (32) ; differentiation of neuroblastoma cells by RA or thyroid hormone is accompanied by p27 up-regulation; and p27 overexpression induces partial neuronal differentiation of mouse neuroblastoma cells (33) .

In this study we have investigated the role of p27 in the process of neuronal differentiation induced by retinoids in EC NT2/D1 cells. We provide straightforward evidence that up-regulation of p27 is a key target of RA signaling in EC cells. In fact, we report that RA enhances p27 expression and induces increased association of p27 with cyclin E/CDK2 complexes and suppresses the activity of these complexes; on the other hand, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by the failure to form neurons or to modify surface antigens and by the lack of neurite outgrowth. Finally, we provide data indicating that the RA-dependent increase in p27 expression results from the reduction in activity of the ubiquitin-proteasome pathway.

Results

Effects of RA on the Growth and Differentiation of NT2/D1 Cells.
Asynchronously proliferating NT2/D1 cells were treated for 4, 7, and 15 days with 10 µM RA; labeled with PI; and analyzed with FACScan. Typically, proliferating NT2/D1 cells showed 44% of cells in the S-phase compartment, whereas on RA treatment, cell growth was arrested, and cells accumulated in G1 (26% of S-phase cells at 4 days of treatment, 16% of S-phase cells at 7 days of treatment, and 8% of S-phase cells at 15 days of treatment, respectively). A representative experiment is reported in Fig. 1ACitation . RA treatment of NT2/D1 cells induces differentiation along the neuronal pathway, with significant morphological changes, outgrowth of neuritis, and modification of surface antigens, as detected by indirect immunofluorescence. Undifferentiated NT2/D1 cells are positive for the SSEA-3 antigen and negative for the neuron-specific A2B5 antigen, whereas RA-treated NT2/D1 cells become negative for SSEA-3 and positive for A2B5. Indirect immunofluorescence analysis of surface antigens in exponentially growing NT2/D1 cells treated for 7 days or 15 days with RA is shown in Fig. 1BCitation . The majority of unstimulated NT2/D1 cells express SSEA-3 but not A2B5. Conversely, RA induced the loss of SSEA-3 expression and the appearance of A2B5 in most cells.



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Fig. 1. Effects of RA on cell cycle and cell cycle-regulatory molecules in NT2/D1 cells. A, asynchronously proliferating NT2/D1 cells were treated for 4, 7, and 15 days with RA (10 µM) and subsequently stained with PI; labeled cells were analyzed with a FACScan using the CELL-FIT program. A representative experiment is shown. B, expression analysis of differentiative markers in exponentially growing cells and in RA-treated NT2/D1 cells (treated for 7 or 15 days, respectively). The results represent the average of three different experiments. C, effects exerted by RA on the expression of CDKs in NT2/D1 cells. D, effects exerted by RA on the expression of cyclins in NT2/D1 cells. E, effects exerted by RA on the expression of CKIs in NT2/D1 cells. Red Ponceau staining of the filters was performed in every experiment to insure uniform protein loading and integrity.

 
Changes in the Levels of Cyclins and CDKs during RA-induced Differentiation.
We investigated the expression of cell cycle regulators in RA-treated NT2/D1 cells. Cell extracts were prepared from NT2/D1 cells exposed to 10 µM RA for 1–7 days and analyzed by Western blot for the expression of cyclins, CDKs, and CKIs. No difference was found in the expression of CDK1, CDK2, CDK4, CDK5, CDK6, and CDK7 (Fig. 1C)Citation . Among the G1 cyclins, expression of cyclin D1 was decreased in RA-treated NT2/D1 cells compared with proliferating NT2/D1 cells. RA did not modify the expression of cyclin E, whereas the expression of cyclin D3, which was expressed at a low level in proliferating cells, increased steadily. The expression of the S-phase cyclin (cyclin A) and the M-phase cyclin (cyclin B) were reduced at 4 and 5 days, respectively (Fig. 1D)Citation .

Expression of CKIs during RA-induced Differentiation.
Western blot analysis of the same cell extracts for the expression of CKIs demonstrated that RA induced an accumulation of p27 protein at 2–3 days after the beginning of RA treatment, with a peak at 7 days (Fig. 1E)Citation . p27 accumulation preceded RA-induced G1 arrest, as indicated by the kinetics of p27 expression relative to cell cycle distribution of NT2/D1 cells, thus providing experimental support to the idea that p27 accumulation is crucial for RA-induced growth arrest of NT2/D1 cells. Our findings that expression of p21 and p16 was increased at 7–10 days of RA treatment (Fig. 1ECitation ; data not shown) suggest a possible role for these inhibitors only in the late events. Conversely, RA did not affect p57 expression (data not shown). Such findings indicated a specific role for p27 in growth inhibition and/or a differentiation commitment of NT2/D1 cells induced by RA.

RA Fails to Induce p27 Expression in NT2-p27AS Cells.
To obtain direct experimental evidence that p27 represents a necessary component of the cellular machinery that transduces RA signaling in NT2/D1 cells, we generated NT2/D1 clones that stably expressed p27 cDNA in the antisense orientation to block the synthesis of the endogenous protein. NT2/D1 cells were transfected with pCMV-p27AS, and several G418-resistant clones were collected. Using a Western blot, we selected two independent NT2-p27AS clones that showed the lowest levels of p27 protein in response to RA (NT2-p27AS-Cl1 and NT2-p27AS-Cl2; Fig. 2ACitation ); however, at a longer exposure time, p27 could also be detected in NT2-p27AS, although at greatly reduced levels.



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Fig. 2. RA fails to induce p27 expression and activity in NT2-p27AS cells. A, top panel, Western blot analysis of p27 in RA-treated NT2/D1 cells or in two different NT2-p27AS clones (AS1 and AS2, respectively). Lower panels, Western blot analysis of cyclin D3, CDK4, cyclin E, and CDK2 in the same cell lysates. B, top panel, association of p27 with CDK2 in NT2/D1 cells or in NT2-p27AS cells treated with RA for 4 or 7 days, as indicated; middle panel, CDK2 level in the immunoprecipitates; bottom panel, CDK2 activity on histone H1. C, top panel, association of p27 with cyclin E in NT2/D1 cells or in NT2-p27AS treated with RA for 4 or 7 days, as indicated; middle panel, cyclin E level in the immunoprecipitates; bottom panel, cyclin E-associated activity on histone H1.

 
Conversely, no difference in the expression of other cell cycle regulators (cyclin D3, cyclin E, CDK2, and CDK4, respectively) was observed in NT2-p27AS cells (Fig. 2A)Citation . We predicted that the increased levels of p27 seen in RA-treated cells should be reflected in an increased amount of this protein associated with cyclin/CDK complexes. As expected, we found significantly higher levels of p27 (5-fold) associated with CDK2 in NT2/D1 cells treated with RA for 4 or 7 days compared with exponentially proliferating cells (Fig. 2Citation B, top panel). Analysis of cyclin E-containing immunocomplexes in proliferating and RA-treated NT2/D1 cells showed that RA increased the levels of p27 associated with cyclin E by 3–4-fold (Fig. 2Citation C, bottom panel). As a result of the increased association of p27 with cyclin E/Cdk2 complexes induced by RA, kinase activity associated with both cyclin E and CDK2 was drastically reduced. Conversely, in NT2-p27AS cells, the RA-dependent increase in the level of p27 associated with cyclin E or CDK2 was greatly reduced, resulting in an increased level of kinase activity in the immunoprecipitates even after 7 days of RA treatment (Fig. 2, B and CCitation , respectively).

RA Inhibits pRB Phosphorylation in NT2/D1 Cells and Suppresses pRB-phosphorylating Activity of CDKs.
The proteins of the pRB family (p105RB, p107, and p130) play a crucial role in growth arrest (34, 35, 36, 37) . pRB is underphosphorylated in G0-early G1 and becomes progressively phosphorylated by several G1 cyclin/CDK complexes during mid- to late G1 (11 , 38) , a change accompanied by an inability to block the G1 to S-phase transition. Western blot analysis of cells committed to differentiate with RA treatment demonstrated that the levels of p130 increased enormously after 7 days of treatment, whereas the levels of pRB were only slightly augmented by RA (Fig. 3A)Citation . However, the hypophosphorylated form of p105 accumulated in response to RA treatment; expression of p107 was detected in neither proliferating nor RA-treated cells. Conversely, in NT2/D1-p27AS cells, pRB phosphorylation was decreased, and accumulation of p130 was completely prevented (Fig. 3)Citation .



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Fig. 3. p27 is necessary for RA-induced growth arrest of NT2/D1 cells. A, Western blot analysis of pRB and p130 in control NT2/D1 or NT2-p27AS cells treated with RA for the indicated times. pRB and phosphorylated pRB (ppRb) are indicated. Red Ponceau staining of the filters was performed in every experiment to insure uniform protein loading and integrity. B, flow cytometry analysis of NT2/D1 or NT2-p27AS cells after treatment with RA for 0 and 15 days. C, BrdUrd incorporation rate of NT2/D1 or NT2-p27AS cells after treatment with RA for 0, 7, and 15 days.

 
p27 Is Necessary for RA-induced Growth Arrest of NT2/D1 Cells.
The two NT2-p27AS clones described above were used to further investigate the molecular mechanism whereby RA induces growth arrest and terminal differentiation in EC cells. No difference was observed between NT2/D1 cells and three different clones obtained using pcDNA-3 vector transfection (NT2-CMV1.1, NT2-CMV1.5, and NT2-CMV1.7); therefore, we will refer to all of them as NT2/D1. NT2-p27AS-Cl1 and NT2-p27AS-Cl2 cells were analyzed for their growth potential by two complementary methodologies: (a) flow cytometry; and (b) measurement of BrdUrd uptake. By flow cytometry, we found that after 15 days of RA treatment, NT2/D1 cells showed only 8% of cells in S phase, whereas RA-treated NT2-p27AS cells showed 20% or 17% of S-phase cells, respectively, as shown in Fig. 3BCitation . Similar results were obtained by measuring the rate of BrdUrd incorporation: 49 ± 6% of untreated NT2/D1 cells incorporated BrdUrd if labeled for 1 h, but only 12 ± 3% or 4 ± 1% of NT2/D1 cells incorporated BrdUrd if treated with RA for 7 days or 15 days, respectively (Fig. 3C)Citation . When cells depleted of p27 (NT2-p27AS-Cl1 and NT2-p27AS-Cl2) were analyzed, we found that on average, 25–28% and 21–24% of cells continued to incorporate BrdUrd after 7 or 15 days of RA treatment, respectively. These results demonstrated that p27 expression is required for G1 arrest induced by RA in NT2/D1 cells.

p27 Is Necessary for RA-induced Differentiation of NT2/D1 Cells.
Subsequently, we investigated whether RA was able to induce morphological or biochemical differentiation in the absence of p27 expression. Cell morphology was investigated by staining polymerized actin with phalloidin. NT2/D1 cells are small polygonal cells. On treatment with RA, the cells become flat and more adherent to the plate, with neuritis extending from the cellular body. NT2-p27AS cells show a morphology similar to that of NT2/D1 cells in the absence of differentiative stimuli. However, when the morphology of NT2-p27AS cells was analyzed after 15–21 days of RA treatment, a different result was obtained:. RA-treated NT2-p27AS cells were generally similar to untreated parental NT2/D1 cells and clearly failed to achieve full terminal differentiation because the outgrowth of neuritis was prevented by depletion of p27 (Fig. 4, A and B)Citation .



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Fig. 4. p27 is necessary for RA-induced differentiation of NT2/D1 cells. A, NT2/D1 or NT2-p27AS cells were seeded onto coverslips, treated with 10 µM RA for 15 days, and subsequently stained with phalloidin to provide evidence of cell morphology. B, neuron formation is impaired in NT2-p27AS cells. The number of neurons in each case was calculated by light microscopy observation by considering neurons to be the cells that have neuritis at least twice as long as the cellular bodies. C, differentiation of NT2/D1 cells by RA is impaired if RA-dependent p27 expression is prevented by antisense methodology. NT2/D1 or NT2-p27AS cells were seeded onto coverslips, treated with 10 µM RA for 15 days, and subsequently stained with anti-SSEA-3 or anti-A2B5 antibodies. D, statistical analysis of SSEA-3 and A2B5 positivity in NT2/D1, NT2-p27AS-Cl1 and NT2/p27AS-Cl2 cells.

 
Differentiation of NT2/D1 cells was also investigated by indirect immunofluorescence analysis of surface markers. Cells were plated onto slides, treated for 21 days with RA, and processed for antigen analysis by indirect immunofluorescence. Neuronal differentiation of NT2/D1 cells induced by RA is reflected by significant modification of surface antigens. Undifferentiated NT2/D1 cells have a SSEA-3+/A2B5- antigenic profile; conversely, RA-treated NT2/D1 cells lose SSEA-3 expression and acquire the expression of A2B5 antigen (Fig. 4Citation C, left column). In contrast with untransfected or vector-transfected cells, RA treatment of NT2-p27AS cells failed to induce down-regulation of SSEA-3 expression and up-regulation of A2B5 expression: the antigenic profile of NT2-p27AS cells remained SSEA-3+/A2B5- (Fig. 4Citation C, right column). Statistical analysis of RA-dependent differentiation of NT2/D1 or NT2-p27AS cells is shown in Fig. 4DCitation .

RA Induced a Block in the Ubiquitin-dependent Proteolysis of p27.
The expression of p27 is essentially regulated at the posttranscriptional level. Two mechanisms in particular have been proposed: (a) modulation of mRNA translation efficiency (29) ; and (b) regulation of protein turnover by a pathway that involves ubiquitination-dependent proteolytic degradation through the 26S proteasome (39) . Ubiquitination of p27 and subsequent degradation require the phosphorylation of a key regulatory residue, threonine 187, by the cyclin E/CDK2 complex (40 , 41) . In this study, we sought to determine the mechanism whereby RA regulated p27 expression in NT2/D1 cells. Northern blot analysis demonstrated that RA only slightly increases in the steady-state levels of p27 mRNA (Fig. 5A)Citation , suggesting that most p27 accumulation after RA treatment was due to a posttranscriptional mechanism. To determine whether the proteasome pathway was involved in the degradation of p27 protein in NT2/D1 cells, cells were treated with two highly specific proteasome inhibitors (the peptide aldheyde LLnL and the inhibitor MG132). As a control, we used the structurally related calpain I inhibitor LLM, which does not act on the 26S proteasome (42) . NT2/D1 cells were treated for 6–12 h with 50 µM LLnL or LLM and 20 µM MG132 and analyzed by Western blot for p27 (Fig. 5B)Citation . As compared with untreated cells, DMSO-treated cells, or cells treated with the same dose of LLM (Fig. 5Citation B, Lanes 1–3, respectively), LLnL treatment resulted in a 3.5-fold increase in the p27 level (Fig. 5Citation B, Lane 4), suggesting that in cycling NT2/D1 cells, the proteasome-dependent pathway takes part in p27 turnover. The same results were obtained with MG132 (Fig. 5Citation B, Lane 5). RA treatment induced the accumulation of a higher molecular weight monoubiquitinated p27. As a complementary approach, we evaluated the ability of extracts from NT2/D1 cells cultured in the absence or presence of RA to degrade recombinant p27 in vitro. Proteasome extracts prepared from undifferentiated cells and from NT2/D1 cells treated for 3 or 6 days with RA were incubated at 37°C with 1 µg of recombinant p27, followed by Western blotting for p27. We found that the extracts derived from cycling NT2/D1 cells degraded exogenous p27 more rapidly than extracts derived cells treated with RA, which suggested that most of RA-induced p27 up-regulation may result from increased p27 protein stability due to the inhibition of p27 degradation. In summary, both in vivo (Fig. 5B)Citation and in vitro (Fig. 5C)Citation experiments indicated that the proteasome pathway was involved in the rapid posttranslational turnover of p27 protein in cycling NT2/D1 cells. Moreover, the results obtained with cell extracts in vitro suggested that RA decreased the turnover of p27 protein. Transfection of FLAG-tagged p27 into NT2/D1 cells in the presence or absence of RA, followed by Western blot analysis with monoclonal anti-p27 antibodies, demonstrated that p27 protein is 2–3-fold less stable in untreated NT2/D1 cells than in NT2/D1 cells treated with RA for 8 days (compare Lanes 1 and 3 in Fig. 6ACitation ). The transfection of FLAG-p27 along with a plasmid encoding ubiquitin did not modify the level of FLAG-p27 in cycling cells or in RA-treated cells (compare Lanes 1and 2 and Lanes 3 and 4 in Fig. 6ACitation ), suggesting that ubiquitin is not rate-limiting for the reduced degradation rate of p27 observed in RA-treated cells.



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Fig. 5. RA induces p27 up-regulation by reducing the proteasome-dependent p27 degradation rate. A, Top panel, Western blot analysis of p27 protein level in exponentially proliferating cells (Lane 1) or RA-treated cells (Lanes 2–7). Red Ponceau staining of the filters was performed in every experiment to insure uniform protein loading and integrity. Middle and bottom panels, Northern blot analysis of p27 in exponentially proliferating (Lane 1) or RA-treated (Lanes 2–7) cells; p27 (middle panel) and glyceraldehyde-3-phosphate dehydrogenase (bottom panel) were used to normalize the amount of RNA loaded. B, effects of LLM, LLnL, and MG132 on the stability of p27 protein. Western blot analysis of p27 protein level in exponentially proliferating cells (Lane 1) and in DMSO (Lane 2)-, LLM (Lane 3)-, LLnL (Lane 4)-, and MG132-treated cells (Lane 5). C, rate of p27 degradation in extracts from proliferating (-) and RA-treated (3 and 6 days, respectively) NT2/D1 cells. One µg of recombinant p27 was incubated at 37°C with 100 µg of proteasome extracts for 0, 6, or 12 h, respectively, and the subsequent Western blotting analysis revealed the amount of intact p27 protein.

 


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Fig. 6. A, Western blot analysis of the steady-state level of FLAG-tagged p27 in solvent or in NT2/D1 cells treated with RA for 6 days. Lane 1, DMSO-treated cells transfected with FLAG-p27; Lane 2, DMSO-treated cells transfected with FLAG-p27 and ubiquitin; Lane 3, cells treated with RA for 6 days and transfected with FLAG-p27; Lane 4, cells treated with RA for 6 days and transfected with FLAG-p27 and ubiquitin; pEGFP was used to normalize for transfection efficiency in all experiments. B, effects of ubiquitin and MG132 on the ubiquitination of wild-type p27 or FLAG-p27T187A mutant. Lane 1 (C), untransfected cells. Lanes 2–5, cells transfected with FLAG- p27T187A in the presence or absence of a plasmid encoding ubiquitin and the proteasome inhibitor MG132, as indicated. Lanes 6–10, cells transfected with wild-type FLAG-p27 in the presence or absence of a plasmid encoding ubiquitin and the proteasome inhibitor MG132, as indicated. All lysates were immunoprecipitated with M2 anti-Flag antibodies (except for those in Lane 6, for which control normal mouse serum was used) and then probed by Western blot with anti-p27 antibodies. Bottom panel, EGFP expression as a control of transfection efficiency. C, effects of RA on p27 ubiquitination. Cells were treated with DMSO or RA for 4 days and then transfected with FLAG-p27. After transfection, DMSO (Lane 1), MG132 (Lane 2), or RA (Lane 3) was added. Proteins were extracted in the presence of 5 mM N-ethyl-maleimide, and p27 ubiquitination was probed with anti-p27 antibodies.

 
Subsequently, we determined whether threonine 187 phosphorylation and consequent ubiquitination were involved in the regulation of p27 protein in NT2/D1 cells. As suggested recently by Montagnoli et al. (40) , degradation of p27 through the proteasome pathway requires threonine 187 phosphorylation-dependent ubiquitination. To determine whether p27 ubiquitination in cycling NT2/D1 cells was dependent on threonine 187, NT2/D1 cells were transfected with FLAG-tagged wild-type p27 or with mutant FLAG-p27187TA in the absence or presence of a plasmid carrying hemagglutinin-tagged ubiquitin. Western blot analysis of cotransfected EGFP was used to normalize transfection efficiency. Cell extracts were prepared, and the appropriate amount of proteins, normalized by the level of transfection efficiency (approximately 400 µg), was immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated proteins were subsequently separated by 12.5% SDS-PAGE and analyzed with monoclonal anti-p27 antibodies (Fig. 6B)Citation . When transfected with FLAG-p27 together with a plasmid encoding ubiquitin, prolonged exposition of the immunoblots also revealed the occurrence of a series of p27-related bands with higher molecular mass, the most prominent of which was of approximately 40 kDa (compare Lanes 7 and 8 in Fig. 6BCitation ). The 40-kDa band has been described as a monoubiquitinated form of p27 (39 , 43) . Ubiquitinated p27 markedly accumulated in the presence of proteasome inhibitors (LLnL or MG132; compare Lanes 7 and 9 or Lanes 8 and 10 in Fig. 6BCitation ), which indicates that in NT2/D1 cells, monoubiquitinated p27 is rapidly degraded unless proteasome activity is inhibited. Conversely, the mutant FLAG-p27187TA could not be ubiquitinated. These results demonstrate that in NT2/D1 cells, p27 degradation occurs by phosphorylation of p27 on threonine 187, ubiquitination, and rapid destruction of the ubiquitinated protein through the 26S proteasome.

Finally, we investigated the effects exerted by RA treatment on the rate of p27 ubiquitination. NT2/D1 cells were treated with DMSO or RA for 4 days, transfected with FLAG-p27, and incubated for 2 additional days with DMSO or RA. The proteasome inhibitor MG132 (20 µM) was added 12 h before cells were collected. Transfection efficiency was determined by immunoblot determination of cotransfected EGFP. Proteins were analyzed by Western blot with anti-FLAG antibodies. Unexpectedly, as shown in Fig. 6CCitation , RA treatment increased the level of ubiquitinated FLAG-p27 as compared with DMSO-treated cells (compare Lanes 2 and 3 in Fig. 6CCitation ). This observation indicates that in NT2/D1 cells, RA does not reduce the ubiquitination of p27 protein but that it does reduce the turnover of ubiquitinated p27, at least at 6 days.

Discussion

In this study, we demonstrate the involvement of the CKI p27 in the growth arrest and neuronal differentiation induced by RA in EC cells. Growth arrest of EC NT2/D1 cells induced by RA is preceded by early accumulation of p27 but not of other CKIs. More direct evidence that causally links RA-dependent growth arrest and p27 protein accumulation is provided by antisense experiments. Inhibition of RA-induced p27 up-regulation in stably transfected p27AS antisense cells results in failure to block cell growth and to progress along the differentiative pathway properly, even after 15 days of RA treatment. In fact, in contrast to NT2/D1 cells, most of which accumulate in the G1 compartment after 7–15 days of RA treatment, we have observed that more than 20% of NT2-p27AS cells are in the S-phase compartment and continue to proliferate in response to RA. Also, the neuronal differentiation program triggered by RA is severely impaired if RA-dependent p27 up-regulation is prevented. In fact, NT2-p27AS cells did not show any morphological sign of neuronal differentiation, which is usually observed in RA-treated NT2/D1 cells. Unlike NT2/D1 cells, which develop neuritis after 2–3 weeks of RA treatment, NT2-p27AS cells maintained a polygonal shape and did not develop neuronal processes even after prolonged exposure to RA. The results obtained from the analysis of differentiation-specific markers were consistent with morphological data. Unlike control cells, NT2-p27AS cells fail to down-regulate expression of SSEA-3 and up-regulate expression of the neuron-specific antigen A2B5 in response to RA.

Retinoids play an important role in neuronal differentiation. However, the molecular basis of RA-dependent signaling has remained elusive thus far. Recently, p27 has been associated with RA-induced growth arrest in SMS-KCNR and LAN-5 human neuroblastoma cell lines, although no functional implications were reported in those studies (43 , 44) . Conversely, our results indicate that p27 up-regulation is required for terminal differentiation signaled by RA. Interestingly, it seems that RA signals preferentially through p27 in cells that differentiate along the neuronal pathway (neuroblastomas and ECs) but not in breast cancer cells (MCF-7; Ref. 45 ). The results reported here allowed us to propose that p27 represents a key regulator of RA signaling in NT2/D1 cells. Accordingly, a previous work from our laboratory showed that in these EC cells, p27 functions at a critical switch point where growth arrest is followed by differentiation (46) . Recently, Spinella et al. (47) have proposed that down-regulation of cyclin D1 may play a role in RA-dependent growth arrest and differentiation of NT2/D1 cells. In fact, constitutive expression of cyclin D1 protein blocked RA-mediated growth arrest and differentiation; moreover, RA receptor {gamma}-deficient NT2/D1 cells (NT2/D1-R1) that do not arrest growth in response to RA showed persistent cyclin D1 overexpression but normal p27 inducibility. These results can be reconciled with the central role of p27 in RA-dependent growth arrest and differentiation of NT2/D1 cells that emerges in this study, if overexpressed cyclin D1 in NT2/D1 cells would not directly activate CDK4–6 but act indirectly by titolating p27 away from cyclinE/CDK2, as is proposed to occur in the titration model (48, 49, 50) .

The mechanism whereby p27 inhibits proliferation in NT2/D1 cells likely involves binding to CDK2-containing complexes with consequent inhibition of the kinase. The inhibition of such G1 CDK activities results in the accumulation of pRB and p130 in their hypophosphorylated, active state, which suggests that the proteins of the retinoblastoma family represent the final end point of RA signaling. Accordingly, accumulation and dephosphorylation of pRB and p130 did not occur in NT2-p27AS cells. It is likely that in NT2/D1 cells, RA blocks degradation of p130 by inhibiting CDK-dependent phosphorylation; conversely, in NT2-p27AS cells, RA is not able to prevent p130 degradation because it fails to suppress CDK activity. This observation is consistent with the notion that in cycling cells, p130 is degraded through a phosphorylation-dependent mechanism, and its accumulation occurs in cells that have definitively left cell cycle and arrest in G0 (38) .

In cycling NT2/D1 cells, p27 levels are regulated posttranslationally. The generally accepted model requires p27 phosphorylation by cyclin E/CDK2 on threonine 187, which allows recognition by proteins that target p27 for ubiquitination and degradation (40 , 51) . Our results indicate that in undifferentiated NT2/D1 cells, the ubiquitin/proteasome pathway is involved in the degradation of p27 protein. The level of p27 protein is markedly up-regulated if NT2/D1 cells are treated with proteasome inhibitors (LLnL and MG132); proteasome-containing extracts derived from cycling NT2/D1 cells show a high degradation rate of exogenous recombinant p27 protein.

The data reported in this work further indicate that in cycling NT2/D1 cells, phosphorylation and subsequent ubiquitination of p27 are involved in p27 turnover. In fact, when cycling NT2/D1 cells are transfected with FLAG-p27 together with a plasmid encoding ubiquitin, prolonged exposition of immunoblots revealed the occurrence of ubiquitinated p27-related bands with higher molecular mass (>40 kDa; Refs. 39 and 43 ). Ubiquitinated p27 accumulates in the presence of proteasome inhibitors (LLnL and MG132), indicating that ubiquitinated p27 is rapidly degraded in untreated NT2/D1 cells, unless proteasome activity is inhibited. Our results also indicate that phosphorylation of threonine 187 is necessary for p27 ubiquitination and degradation because FLAG-p27187TA, a mutant in which the residue threonine 187 has been replaced by alanine, cannot be efficiently ubiquitinated. RA regulates p27 expression by protein stabilization. RA did not induce consistent modification of p27 mRNA levels; proteasome-containing extracts derived from RA-treated cells show a lower degradation rate of exogenous recombinant p27 protein compared with extracts from cycling cells; finally, FLAG-p27 protein is more stable when transiently transfected in RA-treated NT2/D1 cells than in cycling cells. However, RA treatment does not appear to reduce ubiquitination of p27; instead, it reduces the degradation of the ubiquitinated forms.

In conclusion, we demonstrate that p27 represents a key factor in the RA-dependent pathways that regulate growth and terminal differentiation of EC cells. However, additional studies are necessary to clarify the molecular mechanisms whereby RA modulates p27 removal by the 26S proteasome.

Materials and Methods

Cell Culture and Treatment.
The NT2/D1 cell line was grown in DMEM (Sigma Inc.) supplemented with 10% heat-inactivated FCS (Sigma), 4 mM glutamine (Life Technologies, Inc.), 100 units/ml penicillin, and 10 ng/ml streptomycin (Life Technologies, Inc.). RA (Sigma) was solubilized in DMSO and used at a final concentration of 10 µM. Differentiation of the NT2/D1 cell line was performed as described previously (46 , 52) . Cells were plated at a dilution of 1.1 x 106 cells/10-mm culture dish and exposed to 10 µM RA or DMSO for 0, 1, 2, 3, 4, 5, and 7 days. After 7 days in the presence of RA or DMSO, cells were plated at a low dilution and cultured for 1–2 additional weeks to obtain the fully differentiated phenotype.

Flow Cytometric Analysis.
NT2/D1 cells were analyzed for DNA content and expression of cell surface antigens as described previously (46) . Cells were collected and washed in PBS. DNA was stained with PI (50 µg/ml) and analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) interfaced with a Hewlett Packard (Palo Alto, CA) computer. Cell cycle analysis was performed by using the CELL-FIT program (Becton Dickinson; Ref. 46 ). Detection of SSEA-3 or A2B5 monoclonal antibodies was performed as described previously (46) .

RNA Extraction, Northern Blotting, and Hybridization.
Total cellular RNA was isolated from cultured cell lines as described previously (53) . Northern blots were performed as described using nylon Hybond-N membranes (Amersham Pharmacia Biotech) according to the manufacturer’s instructions (54) . All cDNA probes were radiolabeled with a random prime synthesis kit (Amersham Pharmacia Biotech). The probes used in this study are the coding region of human p27 cDNA and the coding region of the human glyceraldehyde-3-phosphate dehydrogenase cDNA obtained by RT-PCR.

Protein Extraction, Western Blotting, and Antibodies.
Cells were scraped in ice-cold PBS and subsequently lysed in ice-cold NP40 lysis buffer [0.5% NP40, 50 mM HEPES (pH 7), 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.5 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin]. In some experiments, 5 mM N-ethyl-maleimide (Sigma) was added to the lysis buffer to preserve ubiquitin-conjugated proteins. Proteins were analyzed on polyacrylamide gel, transferred to nitrocellulose membranes (Hybond-C; Amersham), incubated with specific primary antibodies, and visualized by using enhanced chemiluminescence (Amersham). The antibodies used in this work were obtained from Santa Cruz Biotechnology (anti-p27, C-19; anti-cyclin D3, C16; anti-cyclin D1, HD11; anti-cyclin D2, C-17; anti-cyclin D3, C16; p130, C20), Oncogene Science (anti-cyclin A, AB-2; anti-p21, AB-1), PharMingen (anti-p16, anti-cyclin B1, anti-cyclin D1, anti-cyclin E, HE12; anti-CDK1, A17; anti-pRB, G3-245; anti-cyclin A, BF638; anti-cyclin E, HE67; anti-CDK2 and anti-CDK4), Sigma (anti-FLAG, M2) and Transduction Laboratories (anti-p27, anti-CDK2).

Immunoprecipitation and Kinase Assay.
NT2/D1cells were lysed in NP40 lysis buffer. Proteins (400 µg) were immunoprecipitated with 1–2 µg of the indicated antibodies for 2 h at 4°C and collected on protein A/G-Sepharose (Santa Cruz Biotechnology). Nine-tenths of the immunoprecipitated proteins were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose filters, and incubated with primary antibodies as described above. One-tenth of the immunoprecipitates was resuspended in kinase buffer [20 mM 4-morpholinepropanesulfonic acid (pH 7.2), 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 7.5 mM MgCl2, 50 mM ATP, 1 µCi of [{gamma}-32P]ATP and 5 µg of histone H1] for cyclin E- or CDK2-associated kinase activity or in 1 µg of glutathione S-transferase-pRB 769 (Santa Cruz Biotechnology) for cyclin D3- or CDK4-associated activity and incubated for 15 min at 30°C. Incorporation of radioactive phosphate was determined by using a phosphorimager (GS-525 Bio-Rad) interfaced with a Hewlett Packard computer after SDS-PAGE.

Plasmids and Cell Transfections.
The plasmids encoding human p27 have been described previously (46 , 48) . To obtain stable transfectants in which p27 expression was down-regulated, NT2/D1 cells were transfected with pcDNA-3 or its derivate pCMV-p27AS using N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate liposomal transfection reagents (Roche Biochemical) according to manufacturer’s instructions. After G418 (Life Technologies, Inc.) selection, several resistant clones were picked and analyzed for p27 expression. Among others, two clones positives for p27AS (clones 1 and 2) were used for all described experiments. NT2-CMV clones showed no modification in p27 expression, and the following analyses were conducted using clones NT2-CMV1.1, NT2-CMV1.5, and NT2-CMV1.7.

Immunofluorescence Analysis.
Detection of BrdUrd and surface antigens was carried out essentially as described previously (46 , 52) . Hoechst staining of cell nuclei was performed in each experiment. Fluorescence was analyzed on an epifluorescence microscope Axioplan 2 (Zeiss) able to discriminate between Texas red and Hoechst staining.

In Vitro Degradation of p27 Protein.
In vitro degradation of p27 protein was carried out essentially as described previously (55) . Briefly, NT2/D1 cells were grown for 3 or 7 days in the presence of 10 µM RA or solvent, collected, and frozen immediately at -80°C. Protein extracts were prepared as described previously (55) and incubated (100 µg) with 1 µg of recombinant His-tagged p27 protein. After the indicated times, reactions were stopped by adding 1 volume of 2x Laemmli buffer and loaded onto 12.5% polyacrylamide gel. p27 protein was visualized by using an anti-p27 monoclonal antibody. Quantification of the p27 level was performed by subsequent scanning of films.

Acknowledgments

We are indebted to Dr. P. W. Andrew for kindly providing the differentiation-specific antibodies and Dr. D. Bohman for kindly providing the ubiquitin-hemagglutinin-tagged encoding plasmid.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Associazione Italiana Ricerca sul Cancro and Progetto Finalizzato Biotecnologie of the CNR. A. B., P. B., M. L. M., and B. B. are supported by FIRC fellowships. Back

2 To whom requests for reprints should be addressed, at Oncologia Sperimentale E, Istituto Nazionale Tumori, via M. Semmola, 80131 Naples, Italy. Phone: 081-5903549; Fax: 081-5903838; E-mail: gvigliet{at}tin.it Back

3 The abbreviations used are: RA, retinoic acid; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; pRB, retinoblastoma protein; EGFP, eukaryotic green fluorescent protein; CMV, cytomegalovirus; PI, propidium iodide; EC, embryonal carcinoma; BrdUrd, bromodeoxyuridine; LLnL, N-acetyl-leu-leu-nor leucinal; LLM, N-acetyl-leu-leu-methioninal. Back

Received for publication 6/ 8/00. Revision received 8/30/00. Accepted for publication 8/31/00.

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