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Endocrinology Vol. 144, No. 6 2368-2379
Copyright © 2003 by The Endocrine Society

Microarray and Suppression Subtractive Hybridization Analyses of Gene Expression in Pheochromocytoma Cells Reveal Pleiotropic Effects of Pituitary Adenylate Cyclase-Activating Polypeptide on Cell Proliferation, Survival, and Adhesion

Luca Grumolato, Abdel G. Elkahloun, Hafida Ghzili, David Alexandre, Cédric Coulouarn, Laurent Yon, Jean-Philippe Salier, Lee E. Eiden, Alain Fournier, Hubert Vaudry and Youssef Anouar

Institut Fédératif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology (L.G., H.G., D.A., L.Y., H.V., Y.A.), Institut National de la Santé et de la Recherche Médicale (INSERM) U413, University of Rouen, 76821 Mont-Saint-Aignan, France; Cancer Genetics Branch (A.G.E.), National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892; IFRMP 23, INSERM U519, Faculty of Medicine and Pharmacy (C.C., J.-P.S.), 76183 Rouen, France; Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation (L.E.E.), National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892; and Institut National de la Recherche Scientifique-Institut Armand Frappier (A.F.), University of Quebec, Pointe Claire, Montréal, Canada H9R 1G6

Address all correspondence and requests for reprints to: Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale (INSERM) U413, UA Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic effects on several neuronal, neuroendocrine, and endocrine cells. To gain insight into the pattern of the transcriptional modifications induced by PACAP during cell differentiation, we studied the effects of this neuropeptide on rat pheochromocytoma PC12 cells. We first analyzed the transcriptome of PC12 cells in comparison to that of terminally differentiated rat adrenomedullary chromaffin cells, using a high-density microarray, to identify genes associated with the proliferative phenotype that are possible targets of PACAP during differentiation of sympathoadrenal normal and tumoral cells. We then studied global gene expression in PC12 cells after 48 h of exposure to PACAP, using both cDNA microarray and suppression subtractive hybridization technologies. These complementary approaches resulted in the identification of 75 up-regulated and 70 down-regulated genes in PACAP-treated PC12 cells. Among the genes whose expression is modified in differentiated cells, a vast majority are involved in cell proliferation, survival, and adhesion/motility. Expression changes of most of these genes have been associated with progression of several neoplasms. A kinetic study of the effects of PACAP on some of the identified genes showed that the neuropeptide likely exerts early as well as late actions to achieve the gene expression program necessary for cell differentiation. In conclusion, the results of the present study underscore the pleiotropic role of PACAP in cell differentiation and provide important information on novel targets that could mediate the effects of this neuropeptide in normal and tumoral neuroendocrine cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
DIFFERENTIATION IS A FUNDAMENTAL process necessary for the specification of the various cell phenotypes during development and is a key step of cell growth that allows the transition from proliferating progenitor cells to specialized, functionally oriented cells. Understanding the molecular mechanisms underlying the numerous facets of cell differentiation for a given phenotype can be of utility for the study of not only developmental aspects but also tumorigenic events.

Elucidation of the genetic program that governs differentiation of a cell type can be approached by using in vitro models to gain insight into the molecular events occurring in vivo. The pheochromocytoma PC12 cell line, which originates from a tumor of rat adrenochromaffin cells, has been widely used to decipher the mechanisms of neuroendocrine and neuronal cell differentiation (1, 2). Adrenochromaffin cells are terminally differentiated neuroendocrine cells that derive from neural crest progenitors that also give rise to the sympathetic neurons. Differentiation of PC12 cells can be induced by different trophic factors, including nerve growth factor and glucocorticoids, toward sympathetic and chromaffin-like phenotypes (3, 4).

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid, {alpha}-amidated peptide that regulates multiple functions in the central nervous system and in peripheral tissues via two types of G protein-coupled receptors: a PACAP selective receptor, PAC1-R, and two PACAP/vasoactive intestinal polypeptide mutual receptors, VPAC1-R and VPAC2-R (5, 6, 7). These receptors have been shown to activate different signal transduction pathways that recruit several protein kinases, such as protein kinase A and the MAPK ERK 1/2, which in turn induce or repress transcription of genes associated with homeostasis, growth, and differentiation in various cell types (6, 8, 9, 10). PACAP has been shown to induce growth arrest and to promote neuritic extension in PC12 cells (8, 11, 12), thus offering an opportunity to study the differentiation mechanisms induced by a ligand of G protein-coupled receptors in neuroendocrine cells.

In the adrenal medulla, PACAP has been shown to function as a neurotransmitter to regulate catecholamine, as well as neuropeptide biosynthesis and release in vitro and in vivo, through activation of PAC1-R and downstream signaling cascades in physiological and pathophysiological conditions (13, 14, 15, 16, 17, 18). The presence of PAC1-R has also been demonstrated in pheochromocytomas by receptor autoradiography (19), indicating that PACAP may act on these tumors to influence catecholamine release in vivo, a life-threatening process in patients with pheochromocytoma. In fact, PACAP-like immunoreactivity has also been observed in pheochromocytomas (20), suggesting that an autocrine loop involving PACAP and its receptor may be responsible for a chronic effect of the neuropeptide in this type of tumor. In addition, PACAP may exert trophic and antiapoptotic effects that could influence the progression and differentiation of neoplastic cells, as has been reported in various neuroendocrine tumors (21, 22, 23).

In a recent study aimed at characterizing the phenotype of PACAP-differentiated PC12 cells, we have shown that the neuropeptide elicits a dual neuronal and neuroendocrine differentiation, suggesting that PACAP may represent a trophic factor for sympathoadrenal cells (24). PACAP altered the electrical properties and the expression of genes encoding noradrenergic-determining transcription factors as well as components of the secretory machinery in differentiated cells, indicating that the neuropeptide triggers the transcription of a wide variety of genes to induce cell differentiation. In the present study, we report on the global gene expression changes occurring in PC12 cells that have been differentiated for 48 h by PACAP, using both high-density microarray and suppression subtractive hybridization (SSH) technologies. In addition, validation of PACAP-regulated genes has been performed with a homemade macroarray and by Northern blot and quantitative RT-PCR analyses. These efforts have resulted in the identification of genes and gene families that are candidates for early and late molecular mechanisms underlying cell differentiation induced by PACAP that could occur in normal and tumoral conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals and cell culture
Male Wistar rats (Centre d’Elevage Depré, Saint Doulchard, France) weighing 250–350 g were maintained under controlled conditions of temperature (22 C) under an established photoperiod (lights on from 0700 -1900 h). Rats had free access to laboratory chow (UAR, Epinay-sur-Orge, France) and water. All manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators. PACAP38 was synthesized by the solid phase methodology and purified by HPLC as previously described (25). The identity of the peptide was verified by mass spectrometry. Rat pheochromocytoma PC12 cells were purchased from the European Collection of Cell Culture (Salisbury, Wiltshire, UK). PC12 cells were originally derived from the New England Deaconess Hospital strain of Wistar rats that exhibited a markedly increased incidence of spontaneous pheochromocytoma (1, 26, 27). These cells were maintained in DMEM (Sigma-Aldrich Corp., Saint-Quentin Fallavier, France) supplemented with 10% horse serum (Invitrogen, Cergy Pontoise, France), 5% fetal bovine serum (BioWhittaker Europe, Verviers, Belgium), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich Corp.) at 37 C in 5% CO2. The medium was renewed every 2–3 d. Twenty-four hours after plating, differentiation of PC12 cells was initiated by adding 100 nM PACAP38.

DNA microarrays, hybridization, and data analysis
A glass microarray containing over 15,000 mouse embryonic/placental cDNA probes (28) obtained from the National Institute on Aging (NIA) Mouse 15K cDNA library and corresponding to 15,264 Unigene clusters (for details, see http://lgsun.grc.nia.nih.gov/cDNA/15k.html) was used in this study. Targets were prepared from total RNA that was isolated from rat adrenal medulla or PC12 cells by the method of Chomczynski and Sacchi (29) using the Tri-Reagent (Sigma-Aldrich Corp.). The RNA samples were subsequently purified on RNeasy Mini Spin Columns (QIAGEN, Courtaboeuf, France) and quantified by spectrophotometry. Quality of the RNA was checked by ethidium bromide-staining of the 28S and 18S ribosomal RNA on a formaldehyde-agarose gel. Labeling and hybridization were performed according to standard National Human Genome Research Institute protocols (http://www.nhgri.nih.gov/UACORE/protocols.html). Briefly, 50–100 µg of purified RNA was reverse transcribed with Superscript II reverse transcriptase Rnase H- (Life Technologies, Inc., Gaithersburg, MD) in the presence of either Cy5- or Cy3-deoxyuridine triphosphate (Amersham Pharmacia Biotech, Piscataway, NJ) and oligo(deoxythymidine)12–18 as previously described (30). Microarrays were hybridized with combined Cy5- and Cy3-labeled targets at 65 C overnight in a mix containing 2x Denhardt’s solution, 3.2x saline sodium citrate (SSC), and 0.5% sodium dodecyl sulfate (SDS). The slides were washed at room temperature in 0.5x SSC, 0.1% SDS for 2 min; 0.5x SSC, 0.01% SDS for 2 min; and 0.06x SSC solution for 2 min. The arrays were then scanned (Agilent Technologies, Foster City, CA), and the measured intensities of the red and green fluorescent signals were normalized and filtered through quality control parameters and used to calculate gene expression ratios between the two targets using the IPLab software (Scanalytics, Fairfax, VA). Three independent experiments were performed for each comparative hybridization, and mean values were calculated. PACAP-regulated genes were resequenced, and the identity of the clones was confirmed and updated through comparison with sequences in National Center for Biotechnology Information (NCBI) databases using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).

Rat Genefilter microarrays GF300 and GF301 (Invitrogen) containing more than 10,000 sequence-verified rat cDNA probes obtained from the IMAGE Consortium (Laboratory Integrated Molecular Analysis of Genomes and their Expression, Lawrence Livermore National Laboratory, Livermore, CA) were also used in this study. Target cDNAs from untreated or PACAP-treated PC12 cells were prepared from total RNA after RT as described above, in the presence of [{alpha}-33P]dCTP (Perkin-Elmer Corp., Courtaboeuf, France). The Genefilters were hybridized at 42 C with the 33P-labeled targets in the MicroHyb solution (Invitrogen) supplemented with 1 µg/ml mouse Cot-1 DNA (Invitrogen), 1 µg/ml poly dA (Invitrogen), and 50 µg/ml yeast tRNA (Sigma-Aldrich Corp.). The membranes were washed four times in 2x SSC, 0.1% SDS at room temperature, and twice in 0.1x SSC, 0.1% SDS for 15 min at 50 C. Target cDNAs from each cell condition were simultaneously hybridized to two different Genefilters of the same type, and each Genefilter was then stripped and rehybridized with the opposite target cDNAs to avoid system variability that may be associated with the use of different filters. Images of hybridized Genefilters were obtained using a STORM phosphor imager (Amersham Pharmacia Biotech). The signal intensities of the hybridized probes in a Genefilter were normalized to those of 384 control probes (actin and genomic DNA) that are printed at various areas of the filter, and the consistency of the standardized values in the different hybridizations was assessed by the Pathways 4 software (Invitrogen) before the calculation of gene expression ratios.

SSH
Total RNA was extracted from undifferentiated and PACAP-differentiated PC12 cells as described above, and poly(A)+ RNA was isolated with the PolyATtract mRNA Isolation System (Promega Corp., Charbonnières, France). cDNAs were synthesized from 2 µg of poly(A)+ RNA, and subtractive hybridization was performed using the PCR-Select cDNA subtraction kit (BD Biosciences, Saint-Quentin en Yvelines, France). To isolate PACAP-induced transcripts, cDNAs from PACAP-treated cells were ligated to oligonucleotide linkers and hybridized with excess cDNAs from untreated cells. After hybridization, differentially expressed transcripts were selectively amplified by suppression PCR (31). Amplified cDNAs were introduced into the pCR4-TOPO vector (Invitrogen) and electroporated into DH10B cells to generate a subtractive library. This library was plated, and the plasmids of bacterial lifts were screened to eliminate false positive clones. Briefly, RNA derived from undifferentiated and PACAP-differentiated PC12 cells was reverse transcribed, as described above, in the presence of [{alpha}-32P]dCTP (Amersham Pharmacia Biotech) and used to sequentially hybridize the bacterial lifts at 42 C in a solution containing 50% formamide, 5x SSC, 5x Denhardt’s, 200 µg/ml salmon sperm DNA, 50 µg/ml yeast tRNA, 0.1% SDS, and 50 mM phosphate buffer (pH 6.5). The membranes were washed four times in 2x SSC, 0.1% SDS at room temperature, and twice in 0.1x SSC, 0.1% SDS for 15 min at 50 C. Filters were analyzed using the STORM phosphor imager system, and the images corresponding to hybridization with differentiated or undifferentiated PC12 targets were compared using the Z3 software (Compugen, Jamesburg, NJ) to identify PACAP-regulated clones. Positive clones were identified through sequencing and comparison with sequences in NCBI databases using the BLAST software.

Macroarray preparation and hybridization
Clones identified by microarray and subtractive hybridization analyses were amplified with universal primers and the DyNAzyme EXT DNA polymerase, following the instructions of the manufacturer (Ozyme, Saint-Quentin en Yvelines, France), in a PCRexpress thermal cycler (Hybaid, Paris, France) and used as probes to make a macroarray. The quality of the amplified DNA was checked by migration on a 1% agarose gel. The PCR products contained in a 384-well plate were directly printed on Hybond NX membranes (Amersham Pharmacia Biotech) using a ChipWriter system (Virtek, Waterloo, Canada). These filters were denatured with a 0.4 M NaOH, 0.1 M NaCl solution for 5 min and neutralized with a 40 mM Na2HPO4/NaH2PO4 solution (pH 7.2) for 5 min. The macroarrays were hybridized with target cDNAs derived from untreated or PACAP-treated PC12 cells as described above for the Genefilters. Images of the hybridized macroarrays obtained from the phosphor imager were quantified with the XDotsReader software (Cose, Dugny, France). Hybridization signals were normalized to those of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe that was printed at several locations of the macroarray.

Northern blot analysis
Total RNA was prepared as described above, dissolved in denaturing buffer, heated at 65 C for 15 min, and fractionated on formaldehyde-agarose gels. After staining with ethidium bromide, gels were blotted on Hybond NX membranes (Amersham Pharmacia Biotech) and fixed by UV irradiation. The filters were subsequently hybridized at 42 C with 32P-labeled random primed (Prime-a-Gene Labeling System, Promega Corp.) fragments of inhibitor of DNA binding 3 (Id3), mesoderm specific transcript (Mest), melanoma cell adhesion molecule (Mcam), growth arrest specific 1 (Gas1), chromogranin B (CgB), and brain abundant, membrane-attached signal protein 1 (Basp1) cDNAs in a solution containing 40–50% formamide, 5x SSC, 5x Denhardt’s, 200 µg/ml salmon sperm DNA, 0.1% SDS, 50 mM phosphate buffer (pH 6.5). The membranes were washed as described above for the Genefilters or the macroarrays. Filters were analyzed by using the STORM phosphor imager and the ImageQuant 5.1 software (Amersham Pharmacia Biotech). RNA loading variations were corrected by scanning the ethidium bromide-stained ribosomal RNA using the DensyLab 2.0.5 software (Bioprobe Systems, Montreuil, France).

Quantitative RT-PCR (Q-RT-PCR)
Approximately 1 µg of total RNA extracted as described above was submitted to DNase I (Rnase-free; Promega Corp.) digestion and reverse transcribed using random hexamers pdN6 (Amersham Pharmacia Biotech) and SuperScript II RNase H- reverse transcriptase (Invitrogen). Gene-specific forward and reverse primers were chosen using the Primer Express 2 software (PE Applied Biosystems, Courtaboeuf, France) as follows: 5'-AACTCCCTCAAGATTGTCAGCAA-3' and 5'-GTGGTCATGAGCCCTTCCA-3' for GAPDH; 5'-GCGACACATCGGGAAAGG-3' and 5'-TCGACTCTGCACGAAGATGCT-3' for mothers against decapentaplegic homolog 1 (Madh1); 5'-CAGTTGAAAGAAGAAGGAGTCGTAGA-3' and 5'-AATTCATACTGCTCACTGGTTTGGA-3' for protein tyrosine phosphatase receptor type R (Ptprr). Real-time PCR (Q-RT-PCR) was performed in a premade reaction mix (PE Applied Biosystems) in the presence of the transcribed cDNA and 300 nM of specific primers, using the SYBR green chemistry and an ABI Prism 7000 (PE Applied Biosystems). Relative amounts of Madh1 and Ptprr mRNAs were determined from a standard curve generated using different dilutions of the cDNA and by normalizing against a nonvariable control gene, GAPDH, that was analyzed in parallel on the same RT.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Global analysis revealed differentially expressed genes associated with proliferating pheochromocytoma cells
Because the rat pheochromocytoma PC12 cell line has been originally derived from adrenomedullary chromaffin cells (1, 26), we first compared the transcriptomes of PC12 cells and rat adrenomedullary cells to better understand the effect of PACAP on tumoral cell proliferation and differentiation. We predicted that several genes important for cell growth, survival, and adhesion/motility in this lineage would be identified that could be regulated by trophic factors during differentiation of sympathoadrenal-derived normal or tumoral cells. Gene expression changes between PC12 cells and chromaffin cells were assessed by using a microarray derived from the NIA 15K mouse embryonic cDNA library (28). A mouse microarray was used in this study because a similar rat developmental array was not available. We anticipated that the mouse array made from an embryonic cDNA library could be very useful to identify genes that would be regulated by trophic factors during cell differentiation. Of the 15,264 genes analyzed, 1,048 were differentially expressed by at least 2-fold between the tumoral and normal adrenomedullary chromaffin cells in three independent experiments, using three different RNA preparations from different animals or cell cultures, and three separate hybridizations. The majority of these genes (71.4%) were more highly expressed in PC12 cells compared with adrenomedullary cells, and about 50% were unnamed genes. Because of space limitation, we have arbitrarily chosen to present in this report only named genes that showed a fold change of 2.5 or more (Tables 1Go and 2Go). These genes were classified in functional categories using the Onto-Express V.2 software (32) based on the LocusLink database in NCBI.


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  		Table 1. Named genes overexpressed in PC12 cells vs. rat adrenomedullary cells

 

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  		Table 2. Named genes less expressed in PC12 cells vs. rat adrenomedullary cells

 
Not surprisingly, a vast majority of the named genes that were found to be differentially expressed between PC12 and rat adrenochromaffin cells are associated with proliferation and DNA replication (Tables 1Go and 2Go). In particular, numerous regulators of cell cycle progression were highly expressed in PC12 cells. These include several members of the hexameric mini chromosome maintenance deficient MCM2–7 complex that is required for the onset of the S phase and DNA replication, as well as different cyclins and associated proteins. Of note is the lower expression of cyclin D2 in PC12 compared with adrenomedullary chromaffin cells, in accordance with reports showing an increased expression of this cyclin in certain growth-arrest conditions (33). Several other proteins that interact with components of the cell cycle, including pituitary tumor-transforming 1, prothymosin {alpha}, and thymopoietin, which are implicated in the proliferation of different tumoral cell types (34, 35, 36), also exhibited a higher expression in PC12 cells compared with nontumoral chromaffin cells.

The mRNA of various signaling proteins, e.g. GTPases and related proteins, were present at higher levels in PC12 cells, whereas those of hormonal receptors, e.g. prolactin or melanocortin receptors, were found at higher levels in adrenochromaffin cells. In addition, the expression of several transcription regulators involved in proliferation/differentiation mechanisms, such as members of the high-mobility group protein family, butyrate response factors or GATA-binding proteins, was also altered.

A large group of genes encoding protein processing and apoptosis factors showed a marked expression difference between proliferating PC12 cells and differentiated adrenochromaffin cells (Tables 1Go and 2Go). In this group, several genes encoding ubiquitination and proteasomal factors were more intensely expressed in PC12 cells, reflecting a higher rate of protein degradation in the tumoral cells. On the contrary, genes implicated in protection from oxidative stress, such as glutathione peroxidases, were more highly expressed in chromaffin cells. It should be noted that genes encoding ion transporters like the potassium intermediate/small conductance calcium-activated channel or the selenoprotein P, which were differentially expressed between PC12 and adrenochromaffin cells, may also play a role in cell survival and protection of these cells.

Cytoskeleton and cell matrix/adhesion proteins are involved in a variety of biological responses including remodeling of cell morphology, cell-cell interactions, and cell motility. In this respect, the marked difference in mRNA levels of thymosin-ß 4, an actin-modulating cytoskeletal protein whose expression is related to cell differentiation (37), is especially interesting. Important changes in the expression of numerous matrix and adhesion proteins including galectins, syndecan 1, and embigin, which are implicated in development, cell growth, apoptosis, and differentiation, were observed between PC12 and adrenochromaffin cells. Variations in the expression of these proteins are known to be associated with the aggressiveness and invasiveness of different types of tumors (38, 39, 40).

Although not exhaustive, this comparison of the transcriptomes of PC12 and adrenochromaffin cells provides insights into the genes and gene families whose expression is specifically altered in pheochromocytoma cells, many of which have not previously been described in these cells. The genes identified could represent molecular targets for trophic factors like PACAP to regulate different aspects of growth, survival, and adhesion/motility in physiological and pathophysiological conditions.

Analysis of PACAP-regulated genes in PC12 cells
Treatment of PC12 cells with 10-7 M PACAP for 48 h induced a profound morphological transformation with the appearance of numerous neuritic extensions (Fig. 1Go). Previous studies using similar conditions have shown that PACAP completely suppresses PC12 cell proliferation (41), suggesting that prolonged treatment with PACAP causes growth cessation of the majority of proliferating PC12 cells. We have previously shown that PACAP (10-7 M, 72 h) elicits a dual neuronal and neuroendocrine phenotype as assessed by the effect of the neuropeptide on noradrenergic-specifying transcription factors, cell excitability, and neurotransmitter storage and release machinery in PC12 cells (24). Such actions imply the regulation of a complex program of gene expression during differentiation of PC12 cells.



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  		Figure 1. Effect of PACAP on PC12 cells. Cells were plated at a density of 5 x 105 cells/ml and cultured for 1 d before treatment. The cells were left untreated (Control) or were treated with PACAP (100 nM, 48 h). Scale bar, 50 µm.

 
To determine the molecular events associated with the effect of PACAP on PC12 cells, we compared the transcriptome of cells treated with the neuropeptide for 48 h with that of untreated cells. We hypothesized that, after 2 d, it would be possible to detect changes in the expression of early as well as late genes that could be involved in PC12 cell differentiation under PACAP treatment. To obtain an overall view of the actions of PACAP on PC12 cell gene expression, we used the complementary approaches of high-density microarrays and SSH. We performed microarray gene analysis on the NIA 15K mouse embryonic cDNA library that was used to compare PC12 and chromaffin cell transcriptomes and also on rat random cDNAs from the IMAGE collection that should basically broaden our analysis. We also performed a direct comparison of PC12 cell mRNA populations in PACAP-treated (48 h) and untreated cells using the SSH technique. The rat microarray and SSH should also help to identify PACAP-regulated genes that may fail to hybridize with mouse clones of the NIA library for lack of sufficient sequence homology.

Microarray data analyses were performed by using an average fold change, derived from two independent experiments, of 1.5 or greater and excluding clones that exhibited an incoherent value in any of the different experiments. The ratio limit of 1.5 was used because gene expression changes in this range could be validated in this study by homemade macroarray, Northern blot, and Q-RT-PCR analyses (Fig. 2Go). Indeed, we have performed a macroarray validation of the complete set of cDNAs selected from the NIA microarray and the SSH analysis. Quantification of the hybridization signals confirmed the differential expression of numerous clones between control and PACAP-treated cells (Fig. 2AGo and Tables 3Go and 4Go). As a final verification step, the effect of PACAP on the expression of eight selected genes that represent a range of fold changes was studied individually by using Northern blot and Q-RT-PCR (Fig. 2Go, B and C). In this analysis, clones originally identified from mouse and rat microarrays as well as from SSH screening were examined using specific probes and oligonucleotides. Northern blot analysis confirmed that PACAP up-regulates the expression of the genes encoding the transcription modulator Id3, the cell cycle regulator Gas1, the vesicular protein CgB, and the cell adhesion molecule Mcam, and down-regulates the expression of those encoding Mest and Basp1 with unknown function in PC12 cells (Fig. 2BGo). Using Q-RT-PCR, we confirmed that PACAP significantly stimulates the expression of the transcription factor Madh1 and inhibits that of the signaling protein Ptprr in PC12 cells (Fig. 2CGo). It appears therefore that the regulation by PACAP of the genes characterized in this study, using microarray/SSH and subsequent macroarray analyses, can be confirmed by independent techniques. These results show that the data that are compiled in Tables 3Go and 4Go corresponding to up- and down-regulated genes by PACAP in PC12 cells, respectively, are reliable.



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  		Figure 2. Macroarray, Northern blot, and Q-RT-PCR analyses of PACAP-regulated genes in differentiated PC12 cells. A, RNA from untreated (Control) and PACAP-treated (100 nM, 48 h) PC12 cells was reverse transcribed in the presence of 33P-dCTP and used to hybridize macroarrays containing cDNA probes derived from the NIA microarray and SSH-based library. B, Twenty µg of total RNA from control and PACAP-differentiated (100 nM, 48 h) PC12 cells was analyzed by Northern blot using specific 32P-labeled cDNA probes for Id3, Gas1, CgB, Mcam, Mest, and Basp1. C, Q-RT-PCR analysis of Madh1 and Ptprr gene expression in PC12 cells that were left untreated (Control) or were treated with PACAP (100 nM, 48 h). The corresponding amplicons were electrophoresed at the end of PCR on 3% agarose gel to assess the amplification of a single DNA band. Statistical analysis was performed using the Student’s t test. **, P < 0.01.

 

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  		Table 3. Genes up-regulated by PACAP in PC12 cells

 

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  		Table 4. Genes down-regulated by PACAP in PC12 cells

 
We identified 75 genes whose expression was increased and 70 genes whose expression was decreased in PC12 cells after a 48-h exposure to PACAP. A large majority of the genes identified have not been previously described as PACAP-responsive. Of these, 53 were identified from the mouse microarray, 59 were identified from the rat microarray, and 37 were isolated by SSH. Surprisingly, only four of these genes were commonly identified by more than one technique (Tables 3Go and 4Go). This finding could be attributable to several reasons. First, the genes contained in the two types of microarrays used originate from two quite different sources. The NIA microarray is developmentally oriented because the cDNAs printed were derived from a mouse embryonic library, and we were more successful with this microarray to isolate PACAP-regulated genes involved in proliferation, transcription, and cell signaling. The IMAGE (Invitrogen) microarrays contain random clones, mostly expressed sequence tags (ESTs) that represent more than 80% of the cDNAs printed on these membranes. In fact, most of the PACAP-regulated genes identified from the IMAGE microarrays fall in the unknown category in Tables 3Go and 4Go. These rat ESTs represent 3'-noncoding sequences that are probably more difficult to annotate and to match with other genomes including the mouse genome from which the NIA microarray is derived. The characterization of these ESTs will undoubtedly benefit from the advancement of genomic information. Nevertheless, these latter arrays were used as a complementary material to the NIA microarray to identify additional genes regulated by PACAP. Second, the technologies used for these two kinds of arrays (glass vs. nylon, fluorescence vs. radioactivity, etc.) have quite different sensitivities, and therefore common genes regulated by PACAP should be expressed in the cells at a sufficient level to be detected by both methodologies. Third, the NIA microarray is made of mouse clones, some of which are probably not recognized by targets from rat PC12 cells.

SSH is a different technique that allows a very partial view of the transcriptome compared with microarrays. This approach is interesting, although time-consuming, in that it permits the direct analysis of the transcriptome of the cell model studied and thus the identification of regulated genes that may not be present on defined arrays. Analysis of the 37 genes identified by SSH showed that about half of these (23 genes) are present in the NIA and/or IMAGE (Invitrogen) microarrays used in the present study. Among the latter genes, only four were also found to be changed on the microarrays. These are the genes described above as those that were found changed by more than one technique. This observation implies that microarray analysis probably failed to identify all PACAP-regulated genes, at least in a reproducible manner, although the genes were present on the microarray. For instance, CgB and Madh1, which could not be identified as PACAP-regulated using the NIA or IMAGE microarrays where they are present, respectively, were found changed by SSH, and their variation was confirmed by Northern blot or Q-RT-PCR (Fig. 2Go). Overall, our results underscore the complementarity of these different techniques and the necessity to use various approaches to study global gene expression changes.

PACAP regulates genes controlling cell growth and differentiation
Among the genes found differentially expressed by microarray or SSH analyses in the presence of PACAP, approximately 40% were unnamed genes, and 55% were genes with a known function that can be classified in various categories (Tables 3Go and 4Go). In PC12 cells, PACAP modified the expression of several genes that are known to be implicated in the regulation of cell growth during development or tumorigenesis in various cell types (Table 3Go and 4Go). PACAP is likely to induce PC12 cell growth arrest by inhibiting the expression of cell cycle regulators, including an MCM protein, the cyclin A2, and thymopoietin, as well as transcription effectors such as high-mobility group and GATA proteins. Interestingly, the levels of these mRNAs were higher in PC12 cells than in nonproliferating chromaffin cells (Table 1Go). In addition, three of the four genes up-regulated by PACAP and associated with proliferation (Table 3Go), namely immediate early response 3, Gas1, and B-cell translocation gene 2, are direct targets of the tumor suppressor p53 (42, 43, 44), indicating that PACAP-regulated pathways may be directly involved in the mechanisms of tumorigenesis.

PACAP may influence PC12 cell differentiation by modulating the biosynthesis of signaling factors that are known to control development of a wide variety of tissues. Indeed, we found that PACAP inhibits TGFß2 and increases bone morphogenetic protein 6 mRNA levels, two members of the TGFß family of growth factors that exert pleiotropic effects in nearly all organs, including many roles in neurogenesis (45). Of note is the up-regulation by PACAP of the transcription factors TGFß1-induced transcript 4 (also known as TSC-22) and Madh1 (also known as Smad1), which are important targets of TGF family members. Collectively, these observations indicate that PACAP may recruit the TGF family signaling pathways to induce cell differentiation and homeostasis. It has been shown that PACAP inhibits TGFß1-induced apoptosis in a human pituitary adenoma cell line (46), further supporting the notion that PACAP can modulate the effects of this family of growth factors. It is interesting to note that a recent study that analyzed the short-term effects of PACAP (6 h of treatment) on the transcriptome of PC12 cells has revealed the regulation of various early signaling factors that are probably required to initiate differentiation (47).

The effect of PACAP on PC12 cell differentiation is characterized by the sprouting of neuritic extensions. Three proteins up-regulated by PACAP in these cells, ephrin A2, S-100-related protein, and a serine protease inhibitor (Table 3Go), may be implicated in neurite outgrowth. The expression of the calcium binding S-100 protein is also induced by nerve growth factor in PC12 cells, and transfection of its cDNA has been shown to be able to promote process formation in these cells (48). The receptor tyrosine kinase ephrin A2 is involved in axon guidance and cell migration during embryonic development (49), and the overexpression of this protein is associated with malignancy of some tumors (50). Finally, the effect of serine protease inhibitors on neurite outgrowth has been previously reported in neuroendocrine cells (51). PACAP also regulated the expression of several cytoskeleton proteins that are important effectors of cell morphology remodeling. Most of the PACAP-regulated cytoskeleton proteins were actin-binding proteins that are associated with either polymerization, e.g. calponin (52), or depolymerization, e.g. cofilin (53) and thymosin ß 10 (54), of the actin network. Actin-based motility is critical for both cell migration and extension of neurites (55). In this respect, it is interesting to note that PACAP also stimulated the expression of ras-related homolog (rhoB), a GTPase that regulates actin dynamics to drive neurite extension (55).

PACAP regulates genes controlling cell adhesion
PACAP affected the expression of several genes implicated in cell adhesion and cell-cell contact, which are often altered in tumors. PACAP increased the mRNA levels of laminin receptor 1, which has been shown to be highly expressed in colon carcinoma tissue and lung cancer cells compared with the nontumoral cell counterparts (56, 57). A correlation between the up-regulation of this receptor and the invasive and metastatic phenotype of cancer cells has also been reported (58). The expression of numerous transmembrane glycoproteins including Mcam, attractin, and embigin was increased by PACAP. The expression of Mcam correlates with tumor thickness and metastatic potential of human melanoma cells in nude mice (59). Embigin was found to be more highly expressed in PC12 cells than in adrenochromaffin cells (Table 1Go). Concurrently, PACAP down-regulated the expression of the extracellular matrix proteins glypican 3 and SPARC, which have been recently associated with tumor progression (60, 61). PACAP also decreased the mRNA levels of an ortholog of the mouse claudin 18, a protein of tight junctions (62), implying that the neuropeptide may inhibit cell-cell contacts in differentiating PC12 cells. Altogether, these data show that PACAP controls the expression of genes that play important roles in cell adhesion and motility, suggesting that the neuropeptide may influence these events in physiological and pathophysiological conditions.

PACAP regulates genes controlling cell survival
In accordance with the known antiapoptotic effect of PACAP in different cell types (6, 63), the present study revealed that the neuropeptide regulates several proteins involved in cell death or survival. Thus, PACAP increased the expression of factors that inhibit protein degradation, such as Bcl2-associated athanogene 3 and the low-density lipoprotein receptor-related protein associated protein 1, as well as detoxifying factors that participate in protection against oxidative stress, e.g. peroxiredoxin 5 and thioredoxin reductase (Table 3Go). It is noteworthy that the expression of antioxidant proteins was higher in adrenomedullary cells than in PC12 cells (Table 2Go), suggesting that increased expression of these genes is part of the mechanisms underlying the maintenance and survival of differentiated cells, a process in which PACAP may play a physiological function. Moreover, PACAP down-regulated the expression of a novel apoptosis-inducing protein, named Amida, which has been shown to modulate cell death in the brain (64), as well as a component of the proteasome, the subunit ß type 7. PACAP also decreased the expression of a calcium-activated potassium channel (Table 4Go), which could play an important role in cell shrinkage associated with loss of ions that accompanies apoptosis in many cell types (65).

Different kinetics of the effect of PACAP on PC12 cell gene expression
In the present study, we hypothesized that treatment of PC12 cells for 48 h by PACAP would allow the uncovering of early as well as late effects of the neuropeptide during cell differentiation. To assess the validity of this hypothesis, we have examined post hoc the kinetics of the action of PACAP in PC12 cells on different genes with various functions (Fig. 3Go). We found that PACAP rapidly increases (approximately 5-fold at 6 h of treatment) the gene expression of Gas1, a protein involved in growth arrest, and Id3, a transcription regulator (Fig. 3Go, A and B). The induction of Gas1 was reduced by about 2-fold after 12 h, whereas that of Id3 remained at approximately the same level up to 24 h. These data show that PACAP rapidly induces the expression of genes implicated in cell growth arrest, in line with the results of the study of Vaudry et al. (47). Both Gas1 and Id3 genes were still elevated at 48 h, thus confirming our hypothesis, although we may have missed other early genes that had probably returned to baseline within 48 h. The effect of PACAP on the gene encoding the secretory granule protein CgB exhibited a different pattern because the mRNA levels of this protein gradually increased to reach 6-fold at 72 h of PACAP treatment (Fig. 3CGo). The expression of Basp1, a protein with no known function, also displayed a distinct profile of regulation on PACAP treatment because its mRNA levels were unchanged at 6 h, decreased only at 12 h, and remained inhibited up to 72 h under PACAP exposure (Fig. 3DGo). Altogether, these data show that PACAP likely modifies the expression of various genes with different kinetics to achieve the gene expression program necessary for cell differentiation.



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  		Figure 3. Kinetics of the effects of PACAP on the expression of four representative genes in PC12 cells. Northern blot analysis of 20 µg of total RNA extracted from control cells or cells treated with 100 nM PACAP for the indicated times was performed to assess the mRNA levels of Id3 (A), Gas1 (B), CgB (C), and Basp1 (D). Data are the mean ± SEM of at least three determinations and are expressed relative to control values.

 
Comparison of the genes regulated by PACAP with those differentially expressed between PC12 and adrenochromaffin cells
To uncover potential genes whose expression may represent targets of PACAP during sympathoadrenal differentiation, we performed a clustering of genes regulated by the neuropeptide, as identified on the mouse 15K NIA microarray, with those that are differentially expressed between PC12 and fully differentiated adrenochromaffin cells identified on the same array (Table 5Go). A large proportion of clustered genes belongs to the proliferation and DNA synthesis category, suggesting that the corresponding proteins could represent physiological targets for growth arrest inductive molecules such as PACAP in proliferating sympathoadrenal progenitors. Likewise, the clustering of the transcription regulators Id3 and a LIM only homolog, as well as proteins associated with apoptosis such as the calcium-activated potassium channel and Bcl2-associated athanogene 3, indicates that these factors may also play a role in the differentiation and survival of sympathoadrenal cells. The other clustered genes were mainly those encoding cytoskeleton and cell matrix/adhesion molecules, which are also important effectors of cell differentiation. This comparison shows that several genes regulated by PACAP are also differentially expressed between undifferentiated tumoral chromaffin cells and differentiated adrenomedullary cells, thus arguing for a role of the neuropeptide in the control of cell differentiation in the sympathoadrenal lineage.


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  		Table 5. Genes regulated by PACAP in PC12 cells, and differentially expressed between PC12 and adrenomedullary cells

 
In conclusion, we have made use of two types of microarrays representing a total of 25,000 genes and performed a direct differential screening of PC12 mRNAs by SSH to provide insights into the molecular events orchestrated by PACAP to regulate neuronal and neuroendocrine cell differentiation. The majority of the genes regulated by PACAP in PC12 cells are involved in cell growth, survival, and adhesion, and many of them have been associated with the progression of various tumors. Moreover, several PACAP-regulated genes are differentially expressed between PC12 cells and adrenomedullary chromaffin cells, further supporting the implication of the encoded proteins in the differentiation events that occur in tumoral and nontumoral cells of this lineage. Additional studies are required to demonstrate the occurrence of these mRNAs and the changes in their expression in sympathoadrenal progenitors as well as in human pheochromocytomas or other neuroendocrine tumors. The present study has also revealed the regulation by PACAP of numerous unnamed genes whose characterization will undoubtedly provide novel information on the effects of this important neuropeptide in the nervous and endocrine systems.


   Footnotes
 
This work was supported by grants from INSERM (U413), a Fonds de la Recherche en Santé du Québec-INSERM exchange program, the Conseil Régional de Haute-Normandie, and the Cortico et Medullosurrénale: les Tumeurs (COMETE-2) Network (Programme Hospitalier de Recherche Clinique AOM-02068). L.G. was the recipient of fellowships from the Conseil Régional de la Vallée d’Aoste (Italy), the Fondation pour la Recherche Médicale, and the French Ministry of Foreign Affairs. C.C. was the recipient of fellowships from the French Ministry for Teaching and Research and the Association pour la Recherche sur le Cancer. H.V. was an Affiliated Professor at the Institut National de la Recherche Scientifique-Institut Armand Frappier (Montréal, Canada).

Abbreviations: Basp1, brain abundant, membrane attached signal protein 1; CgB, chromogranin B; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gas1, growth arrest specific 1; Id3, inhibitor of DNA binding 3; Madh1, mothers against decapentaplegic homolog; Mcam, melanoma cell adhesion molecule; Mest, mesoderm specific transcript; PACAP, pituitary adenylate cyclase-activating polypeptide; Ptprr, protein tyrosine phosphatase receptor type R; Q-RT-PCR, quantitative RT-PCR; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; SSH, suppression subtractive hybridization.

Received December 3, 2002.

Accepted for publication February 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Greene LA, Tischler AS 1976 Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73:2424–2428[Abstract/Free Full Text]
  2. Tischler AS, Greene LA 1978 Morphologic and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Lab Invest 39:77–89[Medline]
  3. Vaudry D, Stork PJS, Lazarovici P, Eiden LE 2002 Signaling pathways for PC12 cell differentiation. Science 296:1648–1649[Abstract/Free Full Text]
  4. Anderson DJ, Michelsohn A 1989 Role of glucocorticoids in the chromaffin-neuron developmental decision. Int J Dev Neurosci 7:475–487[CrossRef][Medline]
  5. Arimura A 1998 Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Jpn J Physiol 48:301–331[CrossRef][Medline]
  6. Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H 2000 Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 52:269–324[Abstract/Free Full Text]
  7. Alexandre D, Vaudry H, Grumolato L, Turquier V, Fournier A, Jégou S, Anouar Y 2002 Novel splice variants of type I pituitary adenylate cyclase-activating polypeptide receptor in frog exhibit altered adenylate cyclase stimulation and differential relative abundance. Endocrinology 143:2680–2692[Abstract/Free Full Text]
  8. Deutsch PJ, Sun Y 1992 The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. J Biol Chem 267:5108–5113[Abstract/Free Full Text]
  9. Barrie AP, Clohessy AM, Buensuceso CS, Rogers MV, Allen JM 1997 Pituitary adenylyl cyclase-activating peptide stimulates extracellular signal-regulated kinase 1 or 2 (ERK1/2) activity in a Ras-independent, mitogen-activated protein kinase/ERK kinase 1 or 2-dependent manner in PC12 cells. J Biol Chem 272:19666–19671[Abstract/Free Full Text]
  10. Sherwood NM, Krueckl SL, McRory JE 2000 The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr Rev 21:619–670[Abstract/Free Full Text]
  11. Hernandez A, Kimball B, Romanchuk G, Mulholland MW 1995 Pituitary adenylate cyclase-activating peptide stimulates neurite growth in PC12 cells. Peptides 16:927–932[CrossRef][Medline]
  12. Lazarovici P, Jiang H, Fink D 1998 The 38-amino-acid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21ras G protein, and pp60c-src cytoplasmic tyrosine kinase. Mol Pharmacol 54:547–558[Abstract/Free Full Text]
  13. Taupenot L, Mahata SK, Wu H, O’Connor DT 1998 Peptidergic activation of transcription and secretion in chromaffin cells. Cis and trans signaling determinants of pituitary adenylyl cyclase-activating polypeptide (PACAP). J Clin Invest 101:863–876[Medline]
  14. Turquier V, Yon L, Grumolato L, Alexandre D, Fournier A, Vaudry H, Anouar Y 2001 Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-existing activator protein-1-like transcription factors. Mol Pharmacol 60:42–52[Abstract/Free Full Text]
  15. Hamelink C, Tjurmina O, Damadzic R, Young WS, Weihe E, Lee HW, Eiden LE 2002 Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis. Proc Natl Acad Sci USA 99:461–466[Abstract/Free Full Text]
  16. Hamelink C, Lee HW, Chen Y, Grimaldi M, Eiden LE 2002 Coincident elevation of cAMP and calcium influx by PACAP-27 synergistically regulates vasoactive intestinal polypeptide gene transcription through a novel PKA-independent signaling pathway. J Neurosci 22:5310–5320[Abstract/Free Full Text]
  17. Tornøe K, Hannibal J, Børglum Jensen T, Georg B, Rickelt LF, Andreasen MB, Fahrenkrug J, Holst JJ 2000 PACAP-(1–38) as neurotransmitter in the porcine adrenal glands. Am J Physiol 279:1413–1425
  18. Lamouche S, Yamaguchi N 2003 PACAP release from the canine adrenal gland in vivo: functional role in adrenomedullary response to severe hypotension. Am J Physiol 284:588–597
  19. Reubi JC, Läderach U, Waser B, Gebbers JO, Robberecht P, Laissue JA 2000 Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin. Cancer Res 60:3105–3112[Abstract/Free Full Text]
  20. Takahashi K, Totsune K, Murakami O, Sone M, Itoi K, Miura Y, Mouri T 1993 Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in pheochromocytomas. Peptides 14:365–369[CrossRef][Medline]
  21. Zia F, Fagarasan M, Bitar K, Coy DH, Pisegna JR, Wank SA, Moody TW 1995 Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells. Cancer Res 55:4886–4891[Abstract/Free Full Text]
  22. Douziech N, Lajas A, Coulombe Z, Calvo E, Laine J, Morisset J 1998 Growth effects of regulatory peptides and intracellular signaling routes in human pancreatic cancer cell lines. Endocrine 9:171–183[CrossRef][Medline]
  23. Leyton J, Coelho T, Coy DH, Jakowlew S, Birrer MJ, Moody TW 1998 PACAP(6–38) inhibits the growth of prostate cancer cells. Cancer Lett 125:131–139[CrossRef][Medline]
  24. Grumolato L, Louiset E, Alexandre D, Ait-Ali D, Turquier V, Fournier A, Fasolo A, Vaudry H, Anouar Y 2003 PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells. Eur J Neurosci 17:71–82[CrossRef][Medline]
  25. Chartrel N, Tonon MC, Vaudry H, Conlon JM 1991 Primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) and effects of ovine PACAP on frog pituitary. Endocrinology 129:3367–3371[Abstract]
  26. DeLellis RA, Merk FB, Deckers P, Warren S, Balogh K 1973 Ultrastructure and in vitro growth characteristics of a transplantable rat pheochromocytoma. Cancer 32:227–235[CrossRef][Medline]
  27. Yang T, Martignetti JA, Massa SM, Longo FM 2000 Leucocyte common-antigen-related tyrosine phosphatase receptor: altered expression of mRNA and protein in the New England Deaconess Hospital rat line exhibiting spontaneous pheochromocytoma. Carcinogenesis 21:125–131[Abstract/Free Full Text]
  28. Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood WH, Becker KG, Ko MSH 2000 Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc Natl Acad Sci USA 97:9127–9132[Abstract/Free Full Text]
  29. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  30. Allander SV, Nupponen NN, Ringner M, Hostetter G, Maher GW, Goldberger N, Chen Y, Carpten J, Elkahloun AG, Meltzer PS 2001 Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile. Cancer Res 61:8624–8628[Abstract/Free Full Text]
  31. Diatchenko L, Lau YFC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD 1996 Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030[Abstract/Free Full Text]
  32. Khatri P, Draghici S, Ostermeier GC, Krawetz SA 2002 Profiling gene expression using Onto-Express. Genomics 79:266–270[CrossRef][Medline]
  33. Meyyappan M, Wong H, Hull C, Riabowol KT 1998 Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells. Mol Cell Biol 18:3163–3172[Abstract/Free Full Text]
  34. Bernal JA, Luna R, Espina A, Lázaro I, Ramos-Morales F, Romero F, Arias C, Silva A, Tortolero M, Pintor-Toro JA 2002 Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis. Nat Genet 32:306–311[CrossRef][Medline]
  35. Orre RS, Cotter MA, Subramanian C, Robertson ES 2001 Prothymosin {alpha} functions as a cellular oncoprotein by inducing transformation of rodent fibroblasts in vitro. J Biol Chem 276:1794–1799[Abstract/Free Full Text]
  36. Weber PJ, Eckhard CP, Gonser S, Otto H, Folkers G, Beck-Sickinger AG 1999 On the role of thymopoietins in cell proliferation. Immunochemical evidence for new members of the human thymopoietin family. Biol Chem 380:653–660[CrossRef][Medline]
  37. Huff T, Müller CSG, Otto AM, Netzker R, Hannappel E 2001 ß-Thymosins, small acidic peptides with multiple functions. Int J Biochem Cell Biol 33:205–220[CrossRef][Medline]
  38. Mukunyadzi P, Sanderson RD, Fan CY, Smoller BR 2002 The level of syndecan-1 expression is a distinguishing feature in behavior between keratoacanthoma and invasive cutaneous squamous cell carcinoma. Mod Pathol 15:45–49[CrossRef][Medline]
  39. Danguy A, Camby I, Kiss R 2002 Galectins and cancer. Biochim Biophys Acta 1572:285–293[Medline]
  40. Guenette RS, Sridhar S, Herley M, Mooibroek M, Wong P, Tenniswood M 1997 Embigin, a developmentally expressed member of the immunoglobulin super family, is also expressed during regression of prostate and mammary gland. Dev Genet 21:268–278[CrossRef][Medline]
  41. Onoue S, Waki Y, Nagano Y, Satoh S, Kashimoto K 2001 The neuromodulatory effects of VIP/PACAP on PC-12 cells are associated with their N-terminal structures. Peptides 22:867–872[CrossRef][Medline]
  42. Schafer H, Trauzold A, Sebens T, Deppert W, Folsch UR, Schmidt WE 1998 The proliferation-associated early response gene p22/PRG1 is a novel p53 target gene. Oncogene 16:2479–2487[CrossRef][Medline]
  43. Ruaro EM, Collavin L, Del Sal G, Haffner R, Oren M, Levine AJ, Schneider C 1997 A proline-rich motif in p53 is required for transactivation-independent growth arrest as induced by Gas1. Proc Natl Acad Sci USA 94:4675–4680[Abstract/Free Full Text]
  44. Rouault JP, Falette N, Guehenneux F, Guillot C, Rimokh R, Wang Q, Berthet C, Moyret-Lalle C, Savatier P, Pain B, Shaw P, Berger R, Samarut J, Magaud JP, Ozturk M, Samarut C, Puisieux A 1996 Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway. Nat Genet 14:482–486[CrossRef][Medline]
  45. Massagué J 1998 TGF-ß signal transduction. Annu Rev Biochem 67:753–791[CrossRef][Medline]
  46. Oka H, Jin L, Kulig E, Scheithauer BW, Lloyd RV 1999 Pituitary adenylate cyclase-activating polypeptide inhibits transforming growth factor-ß1-induced apoptosis in a human pituitary adenoma cell line. Am J Pathol 155:1893–1900[Abstract/Free Full Text]
  47. Vaudry D, Chen Y, Ravni A, Hamelink C, Elkahloun AG, Eiden LE 2002 Analysis of the PC12 cell transcriptome after differentiation with pituitary adenylate cyclase-activating polypeptide (PACAP). J Neurochem 83:1272–1284[CrossRef][Medline]
  48. Masiakowski P, Shooter EM 1990 Changes in PC12 cell morphology induced by transfection with 42C cDNA, coding for a member of the S-100 protein family. J Neurosci Res 27:264–269[CrossRef][Medline]
  49. Karam SD, Burrows RC, Logan C, Koblar S, Pasquale EB, Bothwell M 2000 Eph receptors and ephrins in the developing chick cerebellum: relationship to sagittal patterning and granule cell migration. J Neurosci 20:6488–6500[Abstract/Free Full Text]
  50. Carles-Kinch K, Kilpatrick KE, Stewart JC, Kinch MS 2002 Antibody targeting of the EphA2 tyrosine kinase inhibits malignant cell behavior. Cancer Res 62:2840–2847[Abstract/Free Full Text]
  51. Hill RM, Parmar PK, Coates LC, Mezey E, Pearson JF, Birch NP 2000 Neuroserpin is expressed in the pituitary and adrenal glands and induces the extension of neurite-like processes in AtT-20 cells. Biochem J 345:595–601
  52. Kake T, Kimura S, Takahashi K, Maruyama K 1995 Calponin induces actin polymerization at low ionic strength and inhibits depolymerization of actin filaments. Biochem J 312:587–592
  53. Bamburg JR, McGough A, Ono S 1999 Putting a new twist on actin: ADF/cofilins modulate actin dynamics. Trends Cell Biol 9:364–370[CrossRef][Medline]
  54. Border BG, Lin SC, Griffin WS, Pardue S, Morrison-Bogorad M 1993 Alterations in actin-binding ß-thymosin expression accompany neuronal differentiation and migration in rat cerebellum. J Neurochem 61:2104–2114[Medline]
  55. Meyer G, Feldman EL 2002 Signaling mechanisms that regulate actin-based motility processes in the nervous system. J Neurochem 83:490–503[CrossRef][Medline]
  56. Cioce V, Castronovo V, Shmookler BM, Garbisa S, Grigioni WF, Liotta LA, Sobel ME 1991 Increased expression of the laminin receptor in human colon cancer. J Natl Cancer Inst 83:29–36[Abstract/Free Full Text]
  57. Satoh K, Narumi K, Sakai T, Abe T, Kikuchi T, Matsushima K, Sindoh S, Motomiya M 1992 Cloning of 67-kDa laminin receptor cDNA and gene expression in normal and malignant cell lines of the human lung. Cancer Lett 62:199–203[CrossRef][Medline]
  58. Satoh K, Narumi K, Isemura M, Sakai T, Abe T, Matsushima K, Okuda K, Motomiya M 1992 Increased expression of the 67kDa-laminin receptor gene in human small cell lung cancer. Biochem Biophys Res Commun 182:746–752[CrossRef][Medline]
  59. Mills L, Tellez C, Huang S, Baker C, McCarty M, Green L, Gudas JM, Feng X, Bar-Eli M 2002 Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer Res 62:5106–5114[Abstract/Free Full Text]
  60. Filmus J, Selleck SB 2001 Glypicans: proteoglycans with a surprise. J Clin Invest 108:497–501[CrossRef][Medline]
  61. Bradshaw AD, Sage EH 2001 SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J Clin Invest 107:1049–1054[Medline]
  62. Kollmar R, Nakamura SK, Kappler JA, Hudspeth AJ 2001 Expression and phylogeny of claudins in vertebrate primordia. Proc Natl Acad Sci USA 98:10196–10201[Abstract/Free Full Text]
  63. Vaudry D, Gonzalez BJ, Basille M, Pamantung TF, Fontaine M, Fournier A, Vaudry H 2000 The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32. Proc Natl Acad Sci USA 97:13390–13395[Abstract/Free Full Text]
  64. Irie Y, Yamagata K, Gan Y, Miyamoto K, Do E, Kuo CH, Taira E, Miki N 2000 Molecular cloning and characterization of Amida, a novel protein which interacts with a neuron-specific immediate early gene product Arc, contains novel nuclear localization signals, and causes cell death in cultured cells. J Biol Chem 275:2647–2653[Abstract/Free Full Text]
  65. Platoshyn O, Zhang S, McDaniel SS, Yuan JXJ 2002 Cytochrome c activates K+ channels before inducing apoptosis. Am J Physiol 283:C1298–C1305



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