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• W2043741435 abstract "Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G1/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G1 arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells. Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G1/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G1 arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells. nerve growth factor (β-subunit) mitogen-activated protein kinase epidermal growth factor basic fibroblast growth factor phosphate-buffered saline SV40 large T antigen cytomegalovirus sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid bromodeoxyuridine NGF,1 a neurotrophic polypeptide, belongs to a closely related family of neurotrophins composed of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5. These paracrine hormones activate the development, maintenance, and regeneration of neurons in the nervous system (1Levi-Montalcini R. Science. 1987; 237: 1154-1162Crossref PubMed Scopus (2660) Google Scholar). NGF signals the development of sympathetic, sensory, and a population of central nervous system neurons through its high affinity receptor, TrkA. The induction of neuronal differentiation invokes two interrelated cellular processes: progression through the stages of neurite outgrowth and cell cycle arrest (2Liebermann D.A. Hoffman B. Steinman R.A. Oncogene. 1995; 11: 199-210PubMed Google Scholar). The rat pheochromocytoma cell line PC12, derived from a transplantable chromaffin tumor, provides a model system for the NGF-mediated conversion to a neuronal phenotype (3Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4803) Google Scholar). PC12 cells contain both the tyrosine kinase (TrkA) and low affinity (p75NTR) NGF receptors (4Klein R. Jing S.Q. Nanduri V. O'Rourke E. Barbacid M. Cell. 1991; 65: 189-197Abstract Full Text PDF PubMed Scopus (1132) Google Scholar, 5Hosang M. Shooter E.M. EMBO J. 1987; 6: 1197-1202Crossref PubMed Scopus (80) Google Scholar). Differentiation requires the TrkA receptor and proceeds through the Ras/MAPK pathway (6D'Arcangelo G. Halegoua S. Mol. Cell. Biol. 1993; 13: 3146-3155Crossref PubMed Scopus (136) Google Scholar, 7Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (656) Google Scholar). NGF decreases the growth rate of PC12 cells (8Greene L.A. J. Cell Biol. 1978; 78: 747-755Crossref PubMed Scopus (371) Google Scholar) and, in the short term, causes synchronized PC12 cells to accumulate in the G1phase of the cell cycle with a decrease in DNA synthesis (9Rudkin B.B. Lazarovici P. Levi B.Z. Abe Y. Fujita K. Guroff G. EMBO J. 1989; 8: 3319-3325Crossref PubMed Scopus (150) Google Scholar). Continued exposure to NGF arrests the population in G1 with an increased number in the G2/M phase also (10Ignatius M.J. Chandler C.R. Shooter E.M. J. Neurosci. 1985; 5: 343-351Crossref PubMed Google Scholar). Long-term treatment of PC12 cells with NGF promotes terminal differentiation, in which the PC12 cells resemble sympathetic neurons with a cessation of division, increased substratum adherence, neurite extension, and catecholamine synthesis (3Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4803) Google Scholar). The tumor suppressor protein p53 is a DNA-binding phosphoprotein that helps regulate the cell cycle (reviewed in Ref. 11Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar). Overexpression of wild-type p53 causes either G1 cycle arrest (12Lin D. Shields M.T. Ullrich S.J. Appella E. Mercer W.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9210-9214Crossref PubMed Scopus (269) Google Scholar) or apoptosis (2Liebermann D.A. Hoffman B. Steinman R.A. Oncogene. 1995; 11: 199-210PubMed Google Scholar). Inactivation of p53 is a common event in the development of malignancy and occurs in >50% of all human tumors (13Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7400) Google Scholar). Transgenic mice that have the p53 gene disrupted develop normally (14Donehower L.A. Harvey M. Slagle B.L. McArthur M.J. Montgomery Jr., C.A. Butel J.S. Bradley A. Nature. 1992; 356: 215-221Crossref PubMed Scopus (3988) Google Scholar), indicating the dispensability of these genes in normal survival and development. Furthermore, sympathetic and sensory neurons from p53 null mice can survive in the presence of neurotrophins (15Davies A.M. Rosenthal A. Neurosci. Lett. 1994; 182: 112-114Crossref PubMed Scopus (32) Google Scholar). More significantly, however, the neuronal precursors in p53 knockout mice show an enhanced proliferative potential (16Tsukada T. Tomooka Y. Takai S. Ueda Y. Nishikawa S. Yagi T. Tokunaga T. Takeda N. Suda Y. Abe S. Oncogene. 1993; 8: 3313-3322PubMed Google Scholar), supporting a specific role for p53 in mediating an antiproliferative signal to neurons. These experiments implicate the role of p53 and other cell cycle regulators in NGF-mediated growth arrest of neurons and neuronal progenitors. The NGF-mediated cell cycle arrest of PC12 cells is concomitant with the nuclear translocation of p53 in PC12 cells and primary hippocampal neuronal cultures (17Eizenberg O. Faber-Elman A. Gottlieb E. Oren M. Rotter V. Schwartz M. Mol. Cell. Biol. 1996; 16: 5178-5185Crossref PubMed Scopus (180) Google Scholar, 18Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). The importance of this subcellular movement of p53 was also shown by the stable expression of a p53 dominant-negative miniprotein, in which the cytoplasmic sequestration of wild-type p53 correlated with an inhibition of both PC12 cell and oligodendrocyte differentiation (17Eizenberg O. Faber-Elman A. Gottlieb E. Oren M. Rotter V. Schwartz M. Mol. Cell. Biol. 1996; 16: 5178-5185Crossref PubMed Scopus (180) Google Scholar). Progression through the cell cycle has been shown to be governed by the family of cyclin-dependent kinases, their regulatory subunits (the cyclins), and a family of protein inhibitors (19Sherr C.J. Proc. Assoc. Am. Physicians. 1995; 107: 181-186PubMed Google Scholar). In particular, the cyclin-dependent kinase inhibitor p21/WAF1 (20el-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 (7869) Google Scholar) is a direct transcriptional target gene of p53 and plays an important role in p53-dependent growth arrest (21Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1926) Google Scholar, 22el-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. Pietenpol J.A. Burrell M. Hill D.E. Wang Y. Cancer Res. 1994; 54: 1169-1174PubMed Google Scholar). In this study, we investigated the role of p53 in mediating the NGF antimitogenic signal that regulates the cell cycle of PC12 cells. Experiments with PC12 cell lines overexpressing a temperature-sensitive p53 mutant protein (A135V; p53ts) showed that the functional inactivation of p53 undermines NGF-activated cell cycle arrest, whereas neurite outgrowth continues uninhibited. Our results suggest that the closely coupled processes of cell cycle arrest and neuritogenesis share overlapping regulators; however, the wild-type p53 protein is a key coordinator of the NGF-stimulated G1/S phase cell cycle checkpoint in PC12 cells. PC12 cells and the mutant cell line PC12nnr5 (from Dr. Lloyd Greene) were grown and maintained in complete Dulbecco's modified Eagle's medium as described (3Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4803) Google Scholar, 23Woodruff N.R. Neet K.E. Biochemistry. 1986; 25: 7967-7974Crossref PubMed Scopus (9) Google Scholar). Exponentially growing populations of PC12 cells were split and grown on collagen (Vitrogen), poly-l-lysine (Sigma), or poly-l-ornithine (Sigma) plates or coverslips at least 18 h before treatment with NGF (50 ng/ml), epidermal growth factor (EGF; 50 ng/ml), or basic fibroblast growth factor (bFGF; 50 ng/ml) in complete medium. Mouse NGF (β-subunit) was prepared and purified from mouse submaxillary gland as described previously (24Luo Y. Neet K.E. J. Biol. Chem. 1992; 267: 12275-12283Abstract Full Text PDF PubMed Google Scholar). EGF and bFGF were purchased from Sigma. In the immunocytochemical studies, PC12 cells were grown on poly-l-lysine or poly-l-ornithine coverslips and treated with NGF for 6 days, washed with phosphate-buffered saline (PBS), and rapidly fixed in a −20 °C mixture of methanol/acetone or 100% methanol. Naive PC12 cells were analyzed with the agarose overlay method (25Fukui Y. Yumura S. Yumura T.K. Methods Cell Biol. 1987; 28: 347-356Crossref PubMed Scopus (108) Google Scholar), which allows for enhanced visualization of cellular components in round-shaped, blast-like cells. PC12 cells without the agarose overlay produced the same results, but yielded inferior images. For the agarose overlay method, the coverslip with undifferentiated PC12 cells was overlaid with a thin sheet of agarose, fixed in methanol/acetone, permeabilized in 0.5% Nonidet P-40, and incubated with anti-p53 monoclonal antibodies for 2 h at 37 °C. Differentiated PC12 cells were fixed by the same procedure, but without agarose overlay, due to neurite shearing effects. For immunofluorescent visualization, primary antibody staining was followed by staining with goat anti-mouse secondary antibody conjugated to rhodamine or fluorescein (Chemicon International, Inc.). Colorimetric visualization was performed with a murine avidin-biotin-peroxidase complex kit (Oncogene Science Inc.) and diaminobenzidine (Sigma) according to the manufacturers' specifications. Cells were then studied and photographed with a Nikon camera using a Zeiss microscope or a Nikon Diaphot TMD microscope. For all Western blots, cells were harvested, washed, and stored frozen until all time points were collected. Cell lysates were prepared for Western blotting by homogenizing cells with a Dounce homogenizer or sonicator in lysis buffer containing 25 mm Tris, 2.5 mm EDTA, 250 mm sucrose, 1 mm phenylmethylsulfonyl fluoride, 0.5 units/ml aprotinin, and 1 μg/ml leupeptin (26Sladek T.L. Cell Proliferation. 1996; 29: 579-588Crossref PubMed Scopus (6) Google Scholar). For whole cell lysates, protein concentration was quantitated with the BCA reagent (Pierce) or Bradford reagent (Bio-Rad), and equal amounts of total protein (60–80 μg) were loaded onto SDS-polyacrylamide gel lanes. For the nuclear localization studies, lysates were fractionated into nuclear and cytoplasmic fractions (27Fields A.P. Pettit G.R. May W.S. J. Biol. Chem. 1988; 263: 8253-8260Abstract Full Text PDF PubMed Google Scholar); nuclear pellets were washed at least twice to remove all traces of the cytoplasmic fraction; nuclei were solubilized in sample buffer; and an equal number of nuclei were loaded per lane for SDS-polyacrylamide electrophoresis (18Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). After electrophoresis, proteins were blotted onto nitrocellulose (Schleicher & Schüll) or Immobilon (Bio-Rad), and membranes were blocked with 10% nonfat dry milk for 1 h to overnight. Primary antibody incubation was performed in 5% nonfat dry milk or 3% bovine serum albumin in PBS with the mouse anti-p53 monoclonal antibody PAb421 (Oncogene Science Inc. or Dr. Arnold Levine) or the rabbit anti-p21/WAF1 antibody cg-397 (Santa Cruz Biotechnology). Washing with PBS containing 0.1% Tween 20 was performed between all subsequent steps. Primary antibody staining was followed by staining with horseradish peroxidase-conjugated horse anti-mouse IgG secondary antibody (Bio-Rad) or horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Kirkegaard & Perry Laboratories) in PBS. All Western blots were visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech) and Fuji XR film. All experiments were repeated three times with similar results. Lanes were scanned in an Amersham Pharmacia Biotech laser densitometer and/or a Bio-Rad Gel Doc 1000 to estimate relative levels (18Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). Dr. Moshe Oren provided a temperature-sensitive murine p53 mutant cDNA encoding a valine at amino acid 135 (p53ts) (28Ginsberg D. Michael-Michalovitz D. Oren M. Mol. Cell. Biol. 1991; 11: 582-585Crossref PubMed Scopus (122) Google Scholar). A 5′-EcoRI to 3′-SmaI fragment from the plasmid pp53-3-1 was used to replace the Tag gene in the retroviral vector linker cytomegalovirus (CMV) Tag (29Sladek T.L. Jacobberger J.W. J. Virol. 1992; 66: 1059-1065Crossref PubMed Google Scholar). The resulting virus (linker CMV p53ts) contained a neomycin phosphotransferase gene encoding resistance to the drug G418. Transcription of neomycin phosphotransferase was driven by the 5′-long terminal repeat of the virus. Transcription of the insert was driven by a human CMV immediate-early promoter (30Boshart M. Weber F. Jahn G. Dorsch-Hasler K. Fleckenstein B. Schaffner W. Cell. 1985; 41: 521-530Abstract Full Text PDF PubMed Scopus (840) Google Scholar) internal to the viral long terminal repeats. The empty retroviral vector control (vector) is structurally identical to linker CMV p53ts and linker CMV Tag, except that it lacks the p53 gene insertion downstream of the CMV promoter. Stable PA317 (31Miller A.D. Buttimore C. Mol. Cell. Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1137) Google Scholar) virus producer lines were produced as described previously (29Sladek T.L. Jacobberger J.W. J. Virol. 1992; 66: 1059-1065Crossref PubMed Google Scholar). Budded viral particles from stable viral producer cell lines were harvested from the culture medium and used to infect PC12 cells plated at 60% confluency on collagen-coated plates for 2 h. The cells were incubated at 37 °C and allowed to recover for 2 days before 10 days of G418 (400 μg/ml; Sigma) selection. A control stable population (PC12(vector)) containing the retroviral vector lacking any insert was created and compared with parental PC12 cells in selected experiments. Infected PC12(p53ts), PC12(Tag), and PC12(vector) cell populations were maintained in regular medium in the absence of selection at the nonpermissive temperature (38.5 °C). Overexpression of p53ts or Tag was confirmed in the PC12(p53ts) and PC12(Tag) cells, respectively, by Western blotting and immunoprecipitation or flow cytometry. PC12(vector) cells expressing the vector control construct were tested for G418 resistance by long-term incubation (4 or more weeks), whereas parallel cultures of uninfected PC12 cells expired completely after 10–14 days. The majority of experiments with the PC12(p53ts) cells were conducted at the nonpermissive temperature (38.5 °C) throughout this study to extinguish endogenous wild-type p53 function. Some control experiments with the PC12(p53ts) cells were performed at the permissive temperature (32.5 °C) to evaluate the properties of the wild-type form of p53ts. XTT is a yellow tetrazolium salt that is cleaved by the mitochondrial dehydrogenases in metabolically active (viable) cells to form an orange formazan dye (Roche Molecular Biochemicals cell proliferation kit). The formazan dye was measured at an optimal visible spectrophotometric range of 450–500 nm with an Ortho Diagnostics Systems AutoReader II. All assays were performed in 96-well tissue culture plates coated with 0.1 mg/ml poly-l-ornithine. Aliquots of 103 to 104 cells were plated, allowed to adhere overnight, and incubated in serum-containing medium with varying concentrations of NGF. PC12 cells were kept at the nonpermissive temperature of 38.5 °C; 50 μl of XTT were added to give a final concentration of 0.3 mg/ml; and the absorbance was measured multiple times over a 4–20-h period. All experiments were repeated a minimum of two times, with assays performed in triplicate. The effects on endogenous wild-type p53 activity were monitored with a p53 response element/luciferase reporter construct that was cotransfected with a β-galactosidase reporter for normalization of transfection efficiency (26Sladek T.L. Cell Proliferation. 1996; 29: 579-588Crossref PubMed Scopus (6) Google Scholar). The p53 reporter plasmid contained a basal hsp70 promoter element upstream of the luciferase gene. The DNA consensus binding elements for p53 are located upstream of the hsp70 element (32Martinou I. Fernandez P.A. Missotten M. White E. Allet B. Sadoul R. Martinou J.C. J. Cell Biol. 1995; 128: 201-208Crossref PubMed Scopus (131) Google Scholar). One p53 consensus sequence has the sequence 5′-GGA CAT GCC CGG GCA TGT C-3′ and is linked with and oriented 3′ to the optimal p53 transactivation sequence that consists of four consensus half-sites with the sequence 5′-ACG TTTGCCT TG CCT GGA CTTGCCTGG CCT TGC CTT-3′. The half-sites are denoted by alternating underlined and boldface letters. This alignment potentially facilitates binding of tetrameric p53 (32Martinou I. Fernandez P.A. Missotten M. White E. Allet B. Sadoul R. Martinou J.C. J. Cell Biol. 1995; 128: 201-208Crossref PubMed Scopus (131) Google Scholar), which is reportedly the optimal transactivation conformation in vivo (33Wang P. Reed M. Wang Y. Mayr G. Stenger J.E. Anderson M.E. Schwedes J.F. Tegtmeyer P. Mol. Cell. Biol. 1994; 14: 5182-5191Crossref PubMed Scopus (135) Google Scholar). The tandem linkage of these sequences increased the sensitivity of the reporter construct in an additive manner 30-fold relative to the base-line control (32Martinou I. Fernandez P.A. Missotten M. White E. Allet B. Sadoul R. Martinou J.C. J. Cell Biol. 1995; 128: 201-208Crossref PubMed Scopus (131) Google Scholar). This p53 response element configuration resembles the in vivo situation, where p53 functions transcriptionally as a tetramer. PC12 cells were plated on poly-l-ornithine-coated tissue culture plates at 60% confluency the day before transfection. Plasmids were packaged in LipofectAMINE or LipofectAMINE plus liposome vehicles and transfected according to the protocol of Life Technologies, Inc. for PC12 cells. Cells were then placed in medium containing serum with or without NGF. Cells were harvested 2–3 days later and lysed in RLB buffer (Promega). Total cell lysates were assayed colorimetrically with a β-galactosidase assay kit (Promega) and for luminescence with a luciferase assay kit (Promega). Luciferase data were normalized relative to overall transfection efficiency as determined by β-galactosidase expression, and data are presented as normalized luciferase units. All transfection experiments were performed three times, with the assays in duplicate for each experiment. Morphological and cellular changes of PC12, PC12(vector), and PC12(p53ts) cells in response to NGF were examined in conjunction with bromodeoxyuridine (BrdUrd) incorporation (10 μm, 1–2 h). BrdUrd labeling was analyzed by both immunocytochemistry and flow cytometry to concurrently measure cell cycle phase lengths and distributions. For immunocytochemical studies, cells were grown on poly-l-ornithine-coated coverslips, washed several times with PBS, fixed in a HCl/ethanol mixture, and stained with a horseradish peroxidase-conjugated anti-BrdUrd antibody (Roche Molecular Biochemicals) as described by the manufacturer. Cell immunostaining was visualized colorimetrically with diaminobenzidine following the protocol of the manufacturer, and then the cells were counterstained with 5 mg/ml of eosin Y (Sigma). Cells were observed and photographed using a Nikon Diaphot TMD microscope with a Nikon camera. For flow cytometric studies, cells were washed with PBS, fixed with 70% ethanol, extracted with 3 n HCl to remove histones, and stained with a fluorescein-conjugated anti-BrdUrd antibody (Becton-Dickinson). Counterstaining with propidium iodide (Sigma) and single cell analysis were performed as described (29Sladek T.L. Jacobberger J.W. J. Virol. 1992; 66: 1059-1065Crossref PubMed Google Scholar). A minimum of 10,000 cells/sample were analyzed using a Coulter Epics Elite ESP cytometer. A region was defined by a line drawn around the BrdUrd-positive cells and the percentages of S phase cells were quantitated using Coulter Epics Elite Multigraph software. A 20-base antisense oligonucleotide that corresponds to 10 bases of the 5′-region and 10 bases of the coding region of rat p53 (5′-TGT GAA TCC TCC ATG ACA GT-3′) was made (cf. Ref. 34Tsuji K. Ogawa K. Mol. Carcinog. 1994; 9: 167-174Crossref PubMed Scopus (18) Google Scholar). A corresponding sense oligodeoxynucleotide was used as a negative control. The antisense and sense oligonucleotides (Genosys Biotechnologies, Inc.) were synthesized with a thiol group at the 3′-end and with fluorescein conjugated at the 5′-end to enable visualization of the cellular uptake of the oligonucleotides by fluorescence microscopy (35Troy C.M. Derossi D. Prochiantz A. Greene L.A. Shelanski M.L. J. Neurosci. 1996; 16: 253-261Crossref PubMed Google Scholar, 36Wagner R.W. Nature. 1994; 372: 333-335Crossref PubMed Scopus (801) Google Scholar). The sulfhydryl groups of both oligonucleotides were coupled to penetratin I (Oncor, Inc.), a 16-residue antennapedia homeodomain peptide, to facilitate uptake and nuclear localization by PC12 cells (35Troy C.M. Derossi D. Prochiantz A. Greene L.A. Shelanski M.L. J. Neurosci. 1996; 16: 253-261Crossref PubMed Google Scholar, 37Le Roux I. Joliot A.H. Bloch-Gallego E. Prochiantz A. Volovitch M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9120-9124Crossref PubMed Scopus (105) Google Scholar, 38Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). SDS-polyacrylamide gel electrophoresis with Coomassie Blue staining demonstrated the efficiency of coupling to the peptide. Antisense oligonucleotides, antisense oligonucleotides with a dithiothreitol-decoupled negative control, or sense oligonucleotides were added at 200 nm to PC12 cells in exponential growth for 2 h. The coupled peptide was translocated across the plasma membrane and localized to the nucleus in this time period, as viewed by fluorescence microscopy. NGF (50 ng/ml) was then added to one plate of each antisense or sense oligonucleotide-containing culture along with other appropriate controls. Since nuclear translocation of p53 has been reported to be an important part of its activation process (17Eizenberg O. Faber-Elman A. Gottlieb E. Oren M. Rotter V. Schwartz M. Mol. Cell. Biol. 1996; 16: 5178-5185Crossref PubMed Scopus (180) Google Scholar, 18Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar), we examined this process further. The p53 protein can exist in two different states (mutant/proliferative or wild-type/antiproliferative) that may differ in conformation or degree of phosphorylation (11Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar, 39Ullrich S.J. Anderson C.W. Mercer W.E. Appella E. J. Biol. Chem. 1992; 267: 15259-15262Abstract Full Text PDF PubMed Google Scholar). Specific monoclonal antibodies (40Harlow E. Crawford L.V. Pim D.C. Williamson N.M. J. Virol. 1981; 39: 861-869Crossref PubMed Google Scholar, 41Yewdell J.W. Gannon J.V. Lane D.P. J. Virol. 1986; 59: 444-452Crossref PubMed Google Scholar) that distinguish between these two conformational states of p53 under nondenaturing conditions were used to monitor subcellular localization by immunocytochemistry. The proliferative p53 form (recognized by PAb240) was localized to the cytoplasm alone (Fig. 1 A), whereas the antiproliferative p53 state (recognized by PAb246) was present in the nucleus as well as the cytoplasm (Fig. 1 B). Thus, we have demonstrated the presence of both conformational forms of p53 in normal PC12 cells with distinct subcellular localizations. Cells stained with the general PAb421 antibody, which binds both wild-type and mutant p53 proteins (42Milne D.M. McKendrick L. Jardine L.J. Deacon E. Lord J.M. Meek D.W. Oncogene. 1996; 13: 205-211PubMed Google Scholar), showed a more intense immunoreactivity, positive in both compartments (Fig. 1 C), similar to data obtained by others (17Eizenberg O. Faber-Elman A. Gottlieb E. Oren M. Rotter V. Schwartz M. Mol. Cell. Biol. 1996; 16: 5178-5185Crossref PubMed Scopus (180) Google Scholar). NGF-differentiated PC12 cells were treated for 6 days and immunostained for p53 forms. The proliferative anti-p53 antibody (PAb240) stained the cytoplasm only and not the nucleus (Fig. 1 D), as in the naive PC12 cells. Both antiproliferative p53-specific PAb246 (Fig.1 E) and common binding PAb421 (Fig. 1 F) antibodies again stained nuclear and cytoplasmic regions. All three monoclonal antibodies revealed qualitative increases in immunoreactive intensity upon differentiation. Negative immunostaining controls such as an unrelated primary antibody or secondary antibody conjugates in the absence of primary antibody produced no staining (data not shown). Hence, these data suggest that the proliferative mutant conformation of the p53 protein stays sequestered within the cytoplasm upon NGF induction, whereas the wild-type species enters the nucleus and mediates G1 phase arrest. Furthermore, these findings are consistent with previous Western blotting studies that demonstrated a quantitative increase in total and nuclear p53 proteins in PC12 cells after NGF activation (18Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar). Since cytoplasmic p53 levels are 5-fold higher than nuclear levels (18Gollapudi L. Neet K.E. J. Neurosci. Res. 1997; 49: 461-474Crossref PubMed Scopus (37) Google Scholar), the nuclear increase yielded only a small total cytoplasmic decrease that would easily escape detection by immunocytochemical techniques. To test the specificity of NGF signaling through p53, other growth factors were examined for their effects on p53 protein levels. bFGF activates neurite outgrowth in PC12 cells, similar to NGF (43Rydel R.E. Greene L.A. J. Neurosci. 1987; 7: 3639-3653Crossref PubMed Google Scholar). Treatment of PC12 cells with bFGF for 1, 3, or 6 days caused a growth arrest, as seen with BrdUrd staining (data not shown), and an increase in p53 protein in the nucleus, similar to that caused by NGF (Fig.2 A). EGF affects many of the same signaling pathways in PC12 cells, but stimulates mitogenesis, not neuritogenesis (44Chao M.V. Cell. 1992; 68: 995-997Abstract Full Text PDF PubMed Scopus (267) Google Scholar, 45Qiu M.S. Green S.H. Neuron. 1991; 7: 937-946Abs" @default.
• W2043741435 created "2016-06-24" @default.
• W2043741435 creator A5004485787 @default.
• W2043741435 creator A5015612274 @default.
• W2043741435 creator A5043581551 @default.
• W2043741435 creator A5065039456 @default.
• W2043741435 date "2000-12-01" @default.
• W2043741435 modified "2023-09-29" @default.
• W2043741435 title "Mediation of Nerve Growth Factor-driven Cell Cycle Arrest in PC12 Cells by p53" @default.
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