Ectopic p21WAF1 Expression Induces Differentiation-specific Cell Cycle Changes in PC12 Cells Characteristic of Nerve Growth Factor Treatment
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23517
ISSN1083-351X
AutoresJoseph A. Erhardt, Randall N. Pittman,
Tópico(s)Epigenetics and DNA Methylation
ResumoNerve growth factor treatment of PC12 cells results in neuronal differentiation, a process accompanied by induction of the Cdk inhibitor p21WAF1. To determine the role of p21 in differentiation, PC12 clones containing an inducible p21 construct were utilized to induce growth arrest. Expression of p21 led to accumulation of cyclins D1 and E and to a decrease in cyclins A and B. Levels of Cdc2 and Cdk4 also decreased after p21 induction. Initially, thymidine incorporation into DNA was dramatically inhibited; however, low levels of incorporation were observed during prolonged p21 expression. Fluorescence-activated cell sorter analysis revealed that this low level of DNA synthesis resulted in the generation of polyploid cells. Results from Western blots were consistent with phosphorylation of p21 protein coincident with the resumption of DNA synthesis. Finally, treatment of p21-arrested populations with epidermal growth factor, a known PC12 mitogen, resulted in neurite extension, a key feature of neuronal differentiation. Overall, cell cycle changes following p21 overexpression in PC12 cells closely mimic distinctive events previously shown to occur during differentiation. These results suggest that the mechanism by which nerve growth factor induces the many cellular changes associated with growth arrest during differentiation is through p21WAF1 induction. Nerve growth factor treatment of PC12 cells results in neuronal differentiation, a process accompanied by induction of the Cdk inhibitor p21WAF1. To determine the role of p21 in differentiation, PC12 clones containing an inducible p21 construct were utilized to induce growth arrest. Expression of p21 led to accumulation of cyclins D1 and E and to a decrease in cyclins A and B. Levels of Cdc2 and Cdk4 also decreased after p21 induction. Initially, thymidine incorporation into DNA was dramatically inhibited; however, low levels of incorporation were observed during prolonged p21 expression. Fluorescence-activated cell sorter analysis revealed that this low level of DNA synthesis resulted in the generation of polyploid cells. Results from Western blots were consistent with phosphorylation of p21 protein coincident with the resumption of DNA synthesis. Finally, treatment of p21-arrested populations with epidermal growth factor, a known PC12 mitogen, resulted in neurite extension, a key feature of neuronal differentiation. Overall, cell cycle changes following p21 overexpression in PC12 cells closely mimic distinctive events previously shown to occur during differentiation. These results suggest that the mechanism by which nerve growth factor induces the many cellular changes associated with growth arrest during differentiation is through p21WAF1 induction. nerve growth factor fluorescence-activated cell sorter isopropyl-β-d-thiogalactopyranoside epidermal growth factor retinoblastoma. A key event during terminal differentiation is a permanent withdrawal from the cell cycle; however, the precise mechanisms governing this process are not completely understood. Much attention has focused on regulation of components known to control progression through cell cycle, including cyclins, Cdk proteins (cyclin-dependent kinases) and cyclin-dependent kinase inhibitors. Cyclins bind to partner Cdk proteins, and it is the kinase activity of these heteromeric protein complexes that positively regulate passage through the cell cycle (for reviews, see Refs. 1Graña X. Reddy E.P. Oncogene. 1995; 11: 211-219PubMed Google Scholar, 2Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar, 3Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar). Important regulators of these kinases are cyclin-dependent kinase inhibitors. These proteins bind directly to cyclins and/or Cdk proteins, decreasing the associated activities. Their expression can lead to cell cycle arrest and may contribute to the permanent inhibition of proliferation during differentiation (4MacLachlan T.K. Sang N. Giordano A. Crit. Rev. Eukaryotic Gene Expression. 1995; 5: 127-156Crossref PubMed Scopus (304) Google Scholar, 5Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar, 6Harper J.W. Elledge S.J. Curr. Opin. Genet. Dev. 1996; 6: 56-64Crossref PubMed Scopus (361) Google Scholar). To understand aspects of neuronal differentiation, specifically to investigate events underlying cell cycle arrest during this process, the rat pheochromocytoma (PC12) cell culture model has frequently been employed (7Greene L. Tischler A. Proc. Natl. Acad. Sci . U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar). Undifferentiated PC12 cells proliferate in the presence of serum; however, addition of nerve growth factor (NGF)1 results in sympathetic-like neuronal differentiation, including complete mitotic arrest (7Greene L. Tischler A. Proc. Natl. Acad. Sci . U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar, 8Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Crossref PubMed Google Scholar). Using this system, certain distinctive cell cycle events have been shown to occur. During differentiation, these cells initially arrest in G1, although DNA synthesis continues at a low level, leading to an increase in the DNA content of individual cells (9Ignatius M.J. Chandler C.R. Shooter E.M. J. Neurosci. 1985; 5: 343-351Crossref PubMed Google Scholar, 10Buchkovich K.J. Ziff E.B. Mol. Biol. Cell. 1994; 5: 1225-1241Crossref PubMed Scopus (61) Google Scholar). At the protein level, cyclin D1 increases, cyclin B decreases, cyclin A decreases or remains unchanged, and cyclin E increases or remains unchanged. The cyclin-dependent kinases Cdc2, Cdk2, and Cdk4 have all been shown to decrease as these cells undergo proliferation arrest (11Dobashi Y. Tetsuhiro K. Matsumine A. Toyoshima K. Akiyama T. J. Biol. Chem. 1995; 270: 23031-23037Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar, 14Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). In addition to the above changes, the cyclin-dependent kinase inhibitor p21WAF1 increases at the protein level following NGF treatment of PC12 cells (11Dobashi Y. Tetsuhiro K. Matsumine A. Toyoshima K. Akiyama T. J. Biol. Chem. 1995; 270: 23031-23037Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar, 14Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). p21 was initially isolated as a p53 response gene (15El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar), and a protein increased during senescence (16Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1317) Google Scholar) and differentiation (17Jiang H. Fisher P.B. Mol. Cell. Differ. 1993; 1: 285-299Google Scholar). This gene product was also cloned based on interactions with other cell cycle components (18Xiong Y. Zhang H. Beach D. Cell. 1992; 71: 505-514Abstract Full Text PDF PubMed Scopus (902) Google Scholar, 19Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (709) Google Scholar, 20Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar). p21 is known to directly inhibit kinase activities of a wide range of cyclin-Cdk complexes, resulting in a general arrest in the cell cycle (for review, see Ref. 21Gartel A.L. Serfas M.S. Tyner A.L. Proc. Soc. Exp. Biol. Med. 1996; 213: 138-149Crossref PubMed Scopus (358) Google Scholar). Increased expression of p21 has been associated with differentiation of numerous cell types in addition to neurons, including myelocytes (22Jiang H. Lin J. Su Z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 23Steinman R.A. Hoffman B. Iro A. Guillouf C. Liebermann D.A. El-Houseini M.E. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar), myoblasts (24Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Crossref PubMed Scopus (365) Google Scholar, 25Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1092) Google Scholar), and keratinocytes (26Missero C. Calautti E. Eckner R. Chin J. Tsai L.H. Livingston D.M. Dotto G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5451-5455Crossref PubMed Scopus (329) Google Scholar). Additionally, in vivo analysis of p21 expression reveals consistently high levels in numerous differentiated tissues, including brain (27Huppi K. Siwarski D. Dosik J. Michieli M. Chedid M. Reed S. Mock B. Givol D. Mushinski J.F. Oncogene. 1994; 9: 3017-3020PubMed Google Scholar, 28Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Crossref PubMed Scopus (1025) Google Scholar). Given these data, an obvious potential mechanism underlying permanent withdrawal from the cell cycle during neuronal differentiation could be increased p21 expression. To study the role of p21 in this process, stable PC12 cell lines were generated containing an inducible p21 construct. In a previous report, this system was utilized to demonstrate that irreversible growth arrest occurs in these cells following induction of p21 (29Erhardt J.A. Pittman R.N. Oncogene. 1998; 16: 443-451Crossref PubMed Scopus (52) Google Scholar). The aim of the present study was to investigate the possibility that p21 expression is responsible for the numerous characteristic cell cycle changes associated with terminal differentiation of PC12 cells following NGF treatment. In this study, induction of p21 resulted in growth arrest accompanied by an increase in cyclin D1 and E expression. At the same time, a decrease in cyclins A and B, Cdc2, and Cdk4 was detected. Immediately after p21 induction, thymidine incorporation into DNA was almost completely inhibited; however, a low level of incorporation resumed during long-term growth arrest. FACS analysis revealed that this low level of incorporation was due to an increase in the DNA content of individual cells, rather than a resumption of proliferation. Western analysis of phosphatase-treated cell lysates suggested that p21 protein was phosphorylated during the time of resumed DNA synthesis; similarly, treatment of PC12 cells with NGF led to the same apparent modification. As a whole, these cell cycle changes are the same as those occurring during NGF-induced differentiation of PC12 cells, consistent with p21WAF1 playing a key role in this process. PC12 cell lines containing an inducible p21WAF1 construct were generated as described previously (29Erhardt J.A. Pittman R.N. Oncogene. 1998; 16: 443-451Crossref PubMed Scopus (52) Google Scholar) using the Lac Switch inducible mammalian expression system (Stratagene, La Jolla, CA). A 3 mm concentration of the lactose analogue isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the culture medium to induce p21 expression, with maximal protein levels reached within 14–18 h. Cells were cultured in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal calf serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin at 37 °C in 5% CO2. Clonal cell lines were cultured in the presence of 200 μg/ml hygromycin B and 300 μg/ml G418 to maintain selection. For differentiation, cells were subcultured onto tissue culture dishes precoated with 50 μg/ml rat tail collagen (Collaborative Biochemical, Bedford, MA) in the presence of 100 ng/ml 2.5 S NGF (Collaborative Biochemical) in normal culture medium. Cells treated with 20 ng/ml epidermal growth factor (EGF) (Collaborative Biochemical) were also subcultured onto collagen-coated dishes in normal medium in the presence or absence of IPTG. Total cellular proteins (50 μg/ml) were separated by SDS-10% polyacrylamide gel electrophoresis and transferred to PolyScreen polyvinylidene difluoride transfer membrane (NEN Life Science Products). Antibodies employed to detect p21, cyclin D1, cyclin E, cyclin A, cyclin B, Cdk4, and Cdc2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). For detection of p21, antibodies generated against the carboxyl-terminal 19 amino acids (p21(C)) or amino-terminal 20 amino acids (p21(N)) were utilized. Immunoreactive signals were detected with horseradish peroxidase-conjugated secondary antibodies (Boehringer Mannheim) using Renaissance Western blot chemiluminescent reagent (NEN Life Science Products). To detect potential phosphorylation events, cells containing p21 induced for 8 days were washed in phosphate-buffered saline and resuspended in lysis buffer containing 100 mm Tris (pH 8.0), 5 mm MgCl2, 100 mm NaCl, 0.1% Triton X-100, 10 μg/ml leupeptin, and 0.5 mmphenylmethylsulfonyl fluoride. For lysates receiving no treatment, 2× Laemmli buffer was added immediately. Other lysates were incubated for 30 min at 30 °C with 0.2 units/μl alkaline phosphatase (Boehringer Mannheim) in the presence or absence of the phosphatase inhibitor β-glycerophosphate (20 mm). Reactions were terminated by the addition of 2× Laemmli buffer. Proliferation was assessed by counting the total number of cells in randomly selected fields after 0 days in the absence (control) or presence of 3 mm IPTG to induce p21 expression (six random fields/experiment). Over subsequent days, cells were counted in the same fields, and the relative cell number represents the ratio of cells on a given day to the number of cells at time 0. An average of 350 cells were counted at time 0 in five individual experiments. Neurite extension was measured in randomly selected fields of PC12 cells plated on collagen. Proliferating and p21-arrested cells were cultured in the absence or presence of 20 ng/ml EGF. 4 days after EGF treatment, individual cells were scored based on the number of neurites extending >20 or 50 μm from the cell body. Approximately 60–90 cells/group were analyzed in each experiment, and the data represent the average of three experiments. Cell were treated in the presence or absence of IPTG to induce p21 expression for the indicated times and then incubated with 2 μCi/ml [3H]thymidine for 6 h. Plates were washed two times in cold phosphate-buffered saline and scraped into microcentrifuge tubes. A fraction of cells was removed for subsequent analysis of protein content. The remaining sample was incubated at 4 °C with 10% trichloroacetic acid, and precipitable material was collected on glass filters using a vacuum manifold apparatus. Relative thymidine incorporation into DNA was determined by scintillation counting. Flow cytometric analysis of DNA content was essentially performed as described previously (10Buchkovich K.J. Ziff E.B. Mol. Biol. Cell. 1994; 5: 1225-1241Crossref PubMed Scopus (61) Google Scholar). Proliferating and p21-arrested populations were lysed in a buffer containing 0.3% Nonidet P-40, 5 mm NaCl, 10 mm trisodium citrate (pH 7.6), and 100 μg/ml DNase-free RNase A. Nuclei were isolated by low speed centrifugation, and the resulting pellet was resuspended in lysis buffer containing 5 μg/ml propidium iodide. DNA content was assessed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA), and the resulting data were analyzed using ModFit software (Version 1.00 for Mac, ModFit Verity Software House, Topsham, ME). Isolated nuclei were utilized in this procedure due to the tendency of PC12 cells to aggregate in solution. The inducible p21 system used in this study was described in a previous report (29Erhardt J.A. Pittman R.N. Oncogene. 1998; 16: 443-451Crossref PubMed Scopus (52) Google Scholar). PC12 clonal cell lines were generated containing p21WAF1 under control of the Lac repressor protein. When the lactose analogue IPTG was added, p21 levels increased in these cells, and this increase was followed by an irreversible cell cycle arrest. Fig. 1 demonstrates the time course of growth arrest in these cells. Shown are cell counts from one clonal line in the presence or absence of IPTG (similar results were seen in six different clonal lines). Cells grown in normal medium continued to proliferate, whereas cells grown in the presence of IPTG underwent a dramatic and complete growth arrest, shown here for up to 12 days. The aim of this study was to characterize the nature of the growth arrest induced by p21 in these cells, in particular, to compare results with changes known to occur during cell cycle arrest following NGF-induced neuronal differentiation of PC12 cells. All experiments in this study were performed in numerous clonal cell lines, with similar results obtained with each line. To examine changes in proteins associated with cell cycle progression (cyclins and Cdk proteins) following ectopic p21 induction, samples were taken prior to IPTG treatment and three days following addition of the inducer. Western blots demonstrated the increase in p21 protein in these cells (Fig. 2). Cyclins D1 and E (normally associated with G1/S phase) also increased. In contrast, the mitotic cyclins (A and B1) decreased during the same time period. Similar to the mitotic cyclins, the cyclin-dependent kinases Cdc2 and Cdk4 also decreased following p21 induction (Fig. 2). Overall, the modulation of cell cycle components following p21 induction is characteristic of changes following NGF-induced differentiation (11Dobashi Y. Tetsuhiro K. Matsumine A. Toyoshima K. Akiyama T. J. Biol. Chem. 1995; 270: 23031-23037Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar, 14Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). To further examine growth arrest, incorporation of tritiated thymidine into DNA was measured. Previously, we have shown that there is an immediate decrease in thymidine incorporation following ectopic p21 induction (29Erhardt J.A. Pittman R.N. Oncogene. 1998; 16: 443-451Crossref PubMed Scopus (52) Google Scholar). It was important to examine this phenomenon over a longer time course than previously described, given that PC12 cells differentiated with NGF have the capacity to incorporate thymidine into DNA, even though the cells are terminally differentiated (9Ignatius M.J. Chandler C.R. Shooter E.M. J. Neurosci. 1985; 5: 343-351Crossref PubMed Google Scholar, 10Buchkovich K.J. Ziff E.B. Mol. Biol. Cell. 1994; 5: 1225-1241Crossref PubMed Scopus (61) Google Scholar). An immediate decrease in thymidine incorporation was seen after incubation with IPTG to induce p21 expression (Fig. 3). In this experiment, incorporation initially decreased to near background levels; however, a subsequent increase in thymidine incorporation was seen after 3–6 days of continued growth arrest, although not to the level of actively proliferating cells (∼15% of the maximum). To interpret this late increase in thymidine incorporation into DNA, FACS analysis of DNA content was performed on proliferating and p21-arrested cells. Induction of p21 initially increased the fraction of cells with a 2n DNA content, whereas the fraction with a 3n or 4n DNA content decreased (Fig. 4 A). However, the pattern began to change after 4 days of treatment, with those cells having a 2n DNA content decreasing and those having a 4nDNA content increasing. A slight increase in cells with a 3nDNA content was also observed over the same time period. In Fig. 4 B, the percentage of cells with a 2n or 4n DNA content is provided for this experiment. In proliferating populations (control), the fraction of cells with a 2n DNA content was 65%. Following induction of p21 for 1 day, this increased to 81%. After 4 days, the fraction of cells with a 2n DNA content began to decrease, falling to 75% and finally to 64% after 6 days. The fraction of cells with a 4n DNA content was 12% in proliferating populations and fell to a low of 8% after 2 days. At later time points, the fraction of cells with a 4n DNA content increased, reaching 12% after 4 days and 22% after 6 days. In combination with cell counts and thymidine incorporation data, FACS analysis indicated that long-term arrest of these cells was accompanied by a resumption of DNA synthesis, leading to the generation of polyploid cells in the absence of cell division, a feature characteristic of NGF-induced differentiation of PC12 cells (9Ignatius M.J. Chandler C.R. Shooter E.M. J. Neurosci. 1985; 5: 343-351Crossref PubMed Google Scholar, 10Buchkovich K.J. Ziff E.B. Mol. Biol. Cell. 1994; 5: 1225-1241Crossref PubMed Scopus (61) Google Scholar). Given these changes in DNA synthesis in the absence of proliferation, levels of p21 protein in arrested cells were examined to determine if any modulation was occurring during the time of renewed thymidine incorporation. Using an antibody directed against the amino terminus of p21 (p21(N)), protein levels were seen to increase following IPTG induction and to remain consistently elevated for up to 8 days of treatment (Fig. 5 A). Interestingly, a different pattern was detected when employing an antibody directed against the carboxyl terminus of p21 (p21(C)). In this case, p21 levels also increased following IPTG induction; however, after ∼3 days, the levels began to decrease. NGF is also known to increase p21 protein levels in PC12 cells during terminal differentiation (11Dobashi Y. Tetsuhiro K. Matsumine A. Toyoshima K. Akiyama T. J. Biol. Chem. 1995; 270: 23031-23037Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar, 14Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar); therefore, p21 expression during differentiation was examined to determine if a similar pattern would be detected when employing these two antibodies. As expected, NGF treatment led to a sustained increase in p21 protein when probed with antibody p21(N) (Fig. 5 B). Similar to the results in the p21-inducible cell lines, probing with antibody p21(C) revealed an initial increase in p21 levels, followed by a decrease. To attempt to understand the differences in antibody recognition of p21, lysates from cells treated with IPTG for 8 days were chosen for further study (due to the lack of p21 protein recognition by p21(C) at this time point). An obvious modification that can regulate antibody recognition is phosphorylation; therefore, lysates were treated with alkaline phosphatase to determine if phosphorylation of p21 contributes to the decreased signal observed with antibody p21(C). Western blots (Fig. 5 C) revealed that recognition of p21 by this antibody was dramatically increased in lysates treated with alkaline phosphatase compared with both control lysates and lysates treated with a combination of alkaline phosphatase and the phosphatase inhibitor β-glycerophosphate. These results suggest that the carboxyl-terminal region of p21 is being phosphorylated during long-term growth arrest. The final experiments conducted were aimed at determining the responsiveness of p21-arrested cells to mitogenic stimuli, specifically the ability of arrested cells to respond to EGF. Undifferentiated PC12 cells typically respond to EGF with an increased rate of proliferation, whereas differentiation of PC12 cells with NGF results in a population refractory to EGF treatment, i.e. no proliferative responses are observed (30Lazarovici P. Oshima M. Shavit D. Shibutani M. Jiang H. Monshipouri M. Fink D. Movsesyan V. Guroff G. J. Biol. Chem. 1997; 272: 11026-11034Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Not surprisingly, there was no increase in the number of cells following p21 induction and treatment with EGF (data not shown). Interestingly, EGF treatment did result in neurite extension, similar to that occurring during neuronal differentiation (Fig. 6). Some short neurites were present in control, EGF-treated, and p21-arrested populations; however, numerous long neuritic processes were present following EGF treatment of p21-arrested cells in comparison with the other populations. Quantitation of the average number of neurites/cell extending at least 20 or 50 μm from the cell body showed that EGF-treated p21-arrested cells (EGF/p21) had significantly more neurites after 4 days than control, EGF-treated, or p21-arrested populations (Fig. 7). In examining neurites greater than 50 μm, EGF-treated p21-arrested cells averaged 0.6 neurites/cell body, compared with 0.003 in control, 0.07 in EGF-treated, and 0.05 in p21-arrested populations (i.e. >9-fold more neurites were present in EGF-treated cells compared with other groups). In these experiments, ∼32% of EGF-treated p21-arrested cells had at least one neurite extending >50 μm, compared with 20 or 50 μm. Data represent means ± S.E. of three independent experiments. *,p < 0.05 compared with all other groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) During terminal differentiation of neurons, cells must stop dividing and permanently withdraw from the cell cycle. One of the mechanisms by which this may be accomplished is increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Treatment of PC12 cells with NGF leads to increased expression of p21 protein (11Dobashi Y. Tetsuhiro K. Matsumine A. Toyoshima K. Akiyama T. J. Biol. Chem. 1995; 270: 23031-23037Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar, 14Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). As levels rise, p21 becomes associated with cyclin D1-Cdk4 and cyclin E-Cdk2 complexes, and these associations are correlated with a decrease in the kinase activities (12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar). In neuroblastoma cell lines, NGF-induced differentiation is also accompanied by elevation of p21, and when this increase is blocked, cells fail to differentiate, leading to apoptotic cell death (31Poluha W. Poluha D.K. Chang B. Crosbie N. Schonhoff C.M. Kilpatrick D.L. Ross A.H. Mol. Cell. Biol. 1996; 16: 1335-1341Crossref PubMed Scopus (225) Google Scholar). Overall, these results suggest a role for p21 in cell cycle withdrawal accompanying terminal neuronal differentiation. Previously, an irreversible arrest in proliferation was observed in PC12 cell lines following ectopic expression of p21 (29Erhardt J.A. Pittman R.N. Oncogene. 1998; 16: 443-451Crossref PubMed Scopus (52) Google Scholar). In the present study, cell cycle changes following p21 induction were characterized in an attempt to determine if p21 may be responsible for numerous cell cycle changes that occur in response to NGF. The first parameters investigated were modulation of components associated with cell cycle progression. Induction of p21 leads to an increase in cyclins D1 and E and to a decrease in cyclins A and B. Decreased Cdk4 and Cdc2 expression is also observed in response to p21. Following treatment of PC12 cells with NGF, it has been consistently shown that levels of Cdc2, Cdk4, Cdk2, and cyclin B decrease (12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13van Grunsven L.A. Billon N. Savatier P. Thomas A. Urdiales J.L. Rudkin B.B. Oncogene. 1996; 12: 1347-1356PubMed Google Scholar), whereas levels of cyclin D1 increase (11Dobashi Y. Tetsuhiro K. Matsumine A. Toyoshima K. Akiyama T. J. Biol. Chem. 1995; 270: 23031-23037Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Yan G. Ziff E.B. J. Neurosci. 1995; 15: 6200-6212Crossref PubMed Google Scholar, 13va
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