Disabled-2 Mediates c-Fos Suppression and the Cell Growth Regulatory Activity of Retinoic Acid in Embryonic Carcinoma Cells
2001; Elsevier BV; Volume: 276; Issue: 50 Linguagem: Inglês
10.1074/jbc.m106158200
ISSN1083-351X
AutoresElizabeth R. Smith, Callinice D. Capo‐chichi, Junqi He, Jennifer L. Smedberg, Dong‐Hua Yang, Amanda Prowse, Andrew K. Godwin, Thomas C. Hamilton, Xiang‐Xi Xu,
Tópico(s)Cancer-related Molecular Pathways
ResumoF9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression. F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression. Disabled-2 fetal bovine serum mitogen-activated protein kinase (Erk, extracellular-signal regulated kinase) Dulbecco's modified Eagle's medium 4-morpholinepropanesulfonic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Disabled-2 (DAB2 1 for the human gene and Dab2 for the protein and gene in other species) is one of the two mammalian orthologs of the Drosophila Disabled that was identified as one of the proteins genetically interacting with Abl kinase in fly neuron development (1Hoffmann F.M. Trends Genet. 1991; 7: 351-355Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 2Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The three spliced forms (p96, p93, and p67) of murine Dab2 cDNA were first isolated as mitogen-responsive phosphoproteins functioning in the CSF-1 signal transduction pathway in macrophages (2Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). DAB2 is thought to be a tumor suppressor in ovarian cancer (3Mok S.C. Wong K.K. Chan R.K. Lau C.C. Tsao S.W. Knapp R.C. Berkowitz R.S. Gynecol. Oncol. 1994; 52: 247-252Abstract Full Text PDF PubMed Scopus (168) Google Scholar, 4Tseng C.P. Ely B.D. Li Y. Pong E.C. Hsieh J.T. Endocrinology. 1998; 139: 3542-3553Crossref PubMed Scopus (82) Google Scholar, 5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar, 6Sheng Z. Sun W. Smith E. Cohen C. Sheng Z. Xu X.X. Oncogene. 2000; 19: 4847-4854Crossref PubMed Scopus (67) Google Scholar). Its expression is lost or greatly diminished in 85% of the breast and ovarian cancers analyzed (5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar), and forced re-expression of Dab2 suppresses cell growth and tumorigenicity (4Tseng C.P. Ely B.D. Li Y. Pong E.C. Hsieh J.T. Endocrinology. 1998; 139: 3542-3553Crossref PubMed Scopus (82) Google Scholar, 6Sheng Z. Sun W. Smith E. Cohen C. Sheng Z. Xu X.X. Oncogene. 2000; 19: 4847-4854Crossref PubMed Scopus (67) Google Scholar, 7Mok S.C. Chan W.Y. Wong K.K. Cheung K.K. Lau C.C. Ng S.W. Baldini A. Colitti C.V. Rock C.O. Berkowitz R.S. Oncogene. 1998; 16: 2381-2387Crossref PubMed Scopus (153) Google Scholar). Gene deletions have been found to account for the loss of DAB2 expression in a small percent of tumors. 2Z. Fazili, Z. Sheng, W. Sun, C. Cohen, L. E. Mendez, I. R. Horowitz, A. K. Godwin, and X. X. Xu, submitted for publication. In vertebrates, retinoic acid plays a role in inducing cell lineage in early embryonic development, and defects in retinoic acid metabolism or exposure may result in abnormal development (9De Luca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (816) Google Scholar, 10Ross S.A. McCaffery P.J. Drager U.C. De Luca L.M. Physiol. Rev. 2000; 80: 1021-1054Crossref PubMed Scopus (744) Google Scholar). The GATA transcription factors are believed to serve as mediators of retinoic acid in the induction of the heart, gut, and hematopoietic systems during development (9De Luca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (816) Google Scholar, 10Ross S.A. McCaffery P.J. Drager U.C. De Luca L.M. Physiol. Rev. 2000; 80: 1021-1054Crossref PubMed Scopus (744) Google Scholar, 11Arceci R.J. King A.A. Simon M.C. Orkin S.H. Wilson D.B. Mol. Cell. Biol. 1993; 13: 2235-2246Crossref PubMed Google Scholar, 12Charron F. Nemer M. Semin. Cell Dev. Biol. 1999; 10: 85-91Crossref PubMed Scopus (209) Google Scholar, 13Zaret K. Dev. Biol. 1999; 209: 1-10Crossref PubMed Scopus (185) Google Scholar). Retinoic acid induces gene expression and differentiation in many cell types in culture and exhibits growth suppressive activity in a wide spectrum of tumor cells. Furthermore, retinoic acid has been used successfully to treat leukemia and has been explored for use in treating other malignancies (14Zhang D. Holmes W.F. Wu S. Soprano D.R. Soprano K.J. J. Cell. Physiol. 2000; 185: 1-20Crossref PubMed Scopus (80) Google Scholar, 15Clarkson B. Cancer Cells. 1991; 3: 211-220PubMed Google Scholar, 16Hansen L.A. Sigman C.C. Andreola F. Ross S.A. Kelloff G.J. De Luca L.M. Carcinogenesis. 2000; 21: 1271-1279Crossref PubMed Google Scholar). In in vitro studies of cultured tumor cells, retinoic acid suppresses cyclin D induction and saturation cell density but does not affect log phase cell growth (17Wu S. Donigan A. Platsoucas C.D. Jung W. Soprano D.R. Soprano K.J. Exp. Cell Res. 1997; 232: 277-286Crossref PubMed Scopus (53) Google Scholar, 18Faria T.N. LaRosa G.J. Wilen E. Liao J. Gudas L.J. Mol. Cell. Endocrinol. 1998; 143: 155-166Crossref PubMed Scopus (47) Google Scholar). One of the several possible mechanisms postulated for the effect of retinoic acid on cell growth inhibition is the suppression of AP-1 activity (19Soprano D.R. Chen L.X. Wu S. Donigan A. Borghaei R.C. Soprano K.J. Oncogene. 1996; 12: 577-584PubMed Google Scholar, 20Lin F. Xiao D. Kolluri S.K. Zhang X. Cancer Res. 2000; 60: 3271-3280PubMed Google Scholar), which is the target of activation of the Ras/MEK (kinase for MAPK or Erk)/MAPK pathway by many mitogens. Retinoic acid also induces the transforming growth factor-β pathway, another route for tumor/growth suppression in some systems (21Han G.R. Dohi D.F. Lee H.Y. Rajah R. Walsh G.L. Hong W. Cohen P. Kurie J.M. J. Biol. Chem. 1997; 272: 13711-13716Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The action of retinoic acid is mediated through nuclear receptors that in turn modulate gene expression (9De Luca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (816) Google Scholar, 22Minucci S. Ozato K. Curr. Opin. Genet. Dev. 1996; 6: 567-574Crossref PubMed Scopus (59) Google Scholar). Although some of the direct transcriptional targets of retinoic acid are known, such as the GATA factors (11Arceci R.J. King A.A. Simon M.C. Orkin S.H. Wilson D.B. Mol. Cell. Biol. 1993; 13: 2235-2246Crossref PubMed Google Scholar) and laminin (23Vasios G.W. Gold J.D. Petkovich M. Chambon P. Gudas L.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9099-9103Crossref PubMed Scopus (250) Google Scholar), the principal retinoic acid-controlled growth regulator(s) has yet to be identified, and the mechanisms for retinoic acid regulation and resistance are as yet not fully understood. One of the remarkable changes in cell properties identified recently is that retinoic acid-induced differentiation of F9 cells accompanies the uncoupling of MAPK activation and c-Fos expression (24Smith E.R. Smedberg J.L. Rula M.E. Hamilton T.C. Xu X.X. J. Biol. Chem. 2001; 276: 32094-32100Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), although the mediators of this retinoic acid-induced alteration have not been identified. In addition, some tumor cells develop resistance to growth suppression by retinoic acid (25Early E. Dmitrovsky E. J. Investig. Med. 1995; 43: 337-344PubMed Google Scholar). Loss of retinoic acid receptors accounts for some cases, but other unidentified mechanisms must exist (19Soprano D.R. Chen L.X. Wu S. Donigan A. Borghaei R.C. Soprano K.J. Oncogene. 1996; 12: 577-584PubMed Google Scholar, 25Early E. Dmitrovsky E. J. Investig. Med. 1995; 43: 337-344PubMed Google Scholar, 26Faria T.N. Mendelsohn C. Chambon P. Gudas L.J. J. Biol. Chem. 1999; 274: 26783-26788Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this study using F9 (retinoic acid-sensitive) and PA-1 (retinoic acid-resistant) teratocarcinoma cell lines, we identified the candidate tumor suppressor Dab2 as a retinoic acid-inducible gene in F9 cells but not in PA-1 cells. Dab2 was found to mediate the retinoic acid effect on cell growth inhibition by suppressing c-Fos induction without altering MAPK activation. Transfection/expression of Dab2 is sufficient for cell growth suppression, suggesting that Dab2 is the major mediator of retinoic acid in cell growth suppression. Moreover, the failure or inability to induce Dab2 may be a mechanism for the resistance of tumor cells to retinoic acid in growth suppression. Retinoic acid (all-trans-, 9-cis-retinoic acid) and β-carotene were purchased from Sigma. Tissue culture supplies were obtained from Fisher. DMEM medium was purchased from Mediatech (Herndon, VA); fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Atlanta, GA); TRIzol reagent, 100× antibiotic-antimycotic solution, LipofectAMINE, and serum-free Opti-MEM I medium were purchased from Life Technologies, Inc.; the ECL Super-Signal West Dura extended duration substrate immunodetection reagents were purchased from Pierce; Hybrisol I hybridization solution came from Intergen (Purchase, NY); positively charged nylon membranes were from Roche Molecular Biochemicals; [α-32P]dCTP was from PerkinElmer Life Sciences. All other general chemicals and supplies including Me2SO, ethanol, isopropanol, and agarose were from Sigma or Fisher and were reagent grade or higher. F9 mouse teratocarcinoma and PA-1 human teratocarcinoma cells were purchased from American Type Culture Collection (ATCC). The PA-1 cells were cultured in DMEM supplemented with 10% FBS and 1× antibiotic-antimycotic solution. F9 cells were cultured on gelatin-coated tissue culture plates in DMEM containing 10% heat-inactivated FBS and 1× antibiotic-antimycotic solution. The plates were coated with an autoclaved 0.1% gelatin solution overnight at 4 °C, then washed three times with phosphate-buffered saline before use. Retinoids were added to cells from a 1 mm stock solution in Me2SO. If it is not specifically stated, all-trans-retinoic acid was used. Control cultures contained an equal volume of Me2SO alone. Usually, retinoic acid was added 24 h after plating of cells. Cell growth was determined by either triplicate counting with a hemacytometer or measured using the MTT assay (Promega). The results of MTT assay agreed well with those from cell counting. Anti-Dab2 antibodies were characterized as previously described (2Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar, 6Sheng Z. Sun W. Smith E. Cohen C. Sheng Z. Xu X.X. Oncogene. 2000; 19: 4847-4854Crossref PubMed Scopus (67) Google Scholar, 27Xu X.X. Yi T. Tang B. Lambeth J.D. Oncogene. 1998; 16: 1561-1569Crossref PubMed Scopus (107) Google Scholar). Anti-Dab2 (p96) monoclonal antibodies were purchased from Transduction Laboratories (Lexington, KY); anti-c-Fos came from Santa Cruz Technology; anti-actin came from Sigma; anti-Erk1/2 and anti-phospho-Erk1/2 came from Cell Signaling Technology, Inc. (Beverly, MA). Immunoblotting was performed according to standard procedures, as described previously (5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar, 6Sheng Z. Sun W. Smith E. Cohen C. Sheng Z. Xu X.X. Oncogene. 2000; 19: 4847-4854Crossref PubMed Scopus (67) Google Scholar, 27Xu X.X. Yi T. Tang B. Lambeth J.D. Oncogene. 1998; 16: 1561-1569Crossref PubMed Scopus (107) Google Scholar). After confirmation of antibody selectivity, in some cases two or more antibodies were used simultaneously in an incubation to detect various molecular weight proteins. Total RNA was isolated from cell monolayers according to the TRIzol method (Life Technologies, Inc.). RNA was separated on 1% agarose gel containing 7% formaldehyde and 20 mm MOPS buffer, transferred to positive-charged nylon membranes using 2× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate) buffer, and fixed by baking. DNA probes were labeled with [α-32P]dCTP using a random prime labeling kit (Amersham Pharmacia Biotech). The hybridization and Northern blotting followed standard procedures as described previously (2Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar). The full-length human DAB2 (28Sheng Z. He J. Sun W. Fazili Z. Smith E.R. Dong F.B. Xu X.X. Genomics. 2000; 70: 381-386Crossref PubMed Scopus (30) Google Scholar) or murine Dab2 (2Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) cDNA was inserted into the pcDNA/zeo (Invitrogen, La Jolla, CA) or pMT-CB6+ eukaryotic expression vectors. Plasmid DNA was purified using Qiagen Maxiprep columns. For transfection, 2 μg of Dab2 or vector plasmid DNA were mixed with 20 μl LipofectAMINE in 1 ml of Opti-MEM and added to PA-1 or F9 cells for 16 h. F9 cells were transfected with mouse Dab2 cDNA, and PA-1 cells were transfected with human Dab2 cDNA. The transfection medium was removed, and fresh DMEM containing 10% FBS was added. After 12 h, transfected cells were cultured in DMEM containing 10% FBS and 300 ng/ml zeomycin for selection of pcDNA/zeo vector or 400 μg/ml G418 for selection of pMT-CB6+ vector. This selection medium was changed every 2 days, and after 10–12 days cloning rings were used to isolate positive clones. Cultures were further expanded and examined for Dab2 expression by Western blotting. F9 cells were also transfected with metallothionein promoter-regulated mouse Dab2 construct in pMT-CB6+ vector, and green fluorescent protein in pMT-CB6+ vector was used as a control. To induce expression, 0.1 mm ZnSO4 was added to the medium for 24–72 h. Cell monolayers were released from plates with 0.25% trypsin, 0.1% EDTA and collected by centrifugation. The cells were then fixed in 70% ethanol at 4 °C, pelleted, and re-suspended in 50 μg/ml propidium iodide in phosphate-buffered saline for 30 min at 4 °C. The stained cells were analyzed by flow cytometry performed on a FACScan equipped with argon-ion laser and analyzed by Cell Quest software (Becton Dickinson). Replication-deficient adenovirus expressing Dab2 p96 or p67 spliced forms or β-galactosidase were produced, purified, and titrated as described previously (6Sheng Z. Sun W. Smith E. Cohen C. Sheng Z. Xu X.X. Oncogene. 2000; 19: 4847-4854Crossref PubMed Scopus (67) Google Scholar). For transfection of F9 or PA-1 cells, 100 multiplicity plaque-forming units of adenovirus were added to the cells in medium with low serum (1% FBS) for 4 h. The cells were then used for further experimental manipulation. Under these conditions, more than 90% of the F9 cells expressed the transfected cDNA, as estimated using adenovirus-expressing β-galactosidase. Dab2, a candidate tumor suppressor, is lost in a wide spectrum of tumor tissues and cultured carcinoma cells (5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar). To evaluate mechanisms for its loss, we examined potential factors that might affect Dab2 expression. Dab1, the human ortholog that is mainly expressed in brain, can be induced by retinoic acid in the embryonic P19 carcinoma cell line (29Howell B.W. Gertler F.B. Cooper J.A. EMBO J. 1997; 16: 121-132Crossref PubMed Scopus (303) Google Scholar) and by thyroid hormone (T3 and T4) (30Sheldon M. Nakajima K. Bernal J. Howell B.W. Curran T. Soriano E. Munoz A. J. Neurosci. 1999; 19: 6979-6993Crossref PubMed Google Scholar). Thus we investigated and found that Dab2 can also be induced by retinoic acid in the mouse embryonic teratocarcinoma F9 cell line, which is widely used as a model for studying effects of retinoic acid in gene transcription and cell differentiation. Another recent report also confirmed the ability of retinoic acid to induce Dab2 expression (31Cho S.Y. Cho S.Y. Lee S.H. Park S.S. Mol. Cell. 1999; 30: 179-184Google Scholar). We found that retinoic acid induces expression of both of the two variably spliced forms of Dab2, p96 and p67 (2Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), in F9 cells. The effect is time (Fig. 1 A)- and dose-dependent (Fig. 1 B). High levels of Dab2 protein were induced after treatment with retinoic acid for 4 days, and as little as 10−8m retinoic acid stimulated Dab2 protein expression. Retinoic acid treatment caused greater induction of the p67 form of Dab2, which differs from the expression pattern of Dab2 isoforms found in other cells in which p96 is generally the major or only isoform (2Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar). The induction of Dab2 by retinoic acid occurs at the transcriptional level, because the Dab2 message RNA is induced in a similar magnitude as the protein (Fig. 1 C). Withdrawal of retinoic acid 4 days after induction did not reverse or decrease Dab2 protein levels (Fig. 1 D), and even a month after retinoic acid removal F9 cells continued to express Dab2 (not shown). These results correlate Dab2 expression with the irreversible endoderm differentiation of F9 cells by retinoic acid treatment. Among the retinoids tested, all-trans-retinoid acid is the most potent in the induction of Dab2 (Fig. 1 E). 9-cis-Retinoic acid can induce Dab2 expression in F9 cells, but the required dosage is about 100 times more than that of all-trans-retinoic acid, andN-(4-hydroxylphenyl)retinamide (fenretinide or 4-HPR) and β-carotene (vitamin A) have no detectable activity (Fig. 1 E). In the PA-1 teratocarcinoma cell line, however, retinoic acid treatment for 4 days did not induce Dab2 expression (Fig. 2 A). PA-1 cells were derived from a human ovarian germ cell tumor (32Zeuthen J. Norgaard J.O. Avner P. Fellous M. Wartiovaara J. Vaheri A. Rosen A. Giovanella B.C. Int. J. Cancer. 1980; 25: 19-32Crossref PubMed Scopus (150) Google Scholar) and are resistant to growth suppression by retinoic acid (19Soprano D.R. Chen L.X. Wu S. Donigan A. Borghaei R.C. Soprano K.J. Oncogene. 1996; 12: 577-584PubMed Google Scholar, 33Taylor D.D. Taylor C.G. Black P.H. Jiang C.G. Chou I.N. Differentiation. 1990; 43: 123-130Crossref PubMed Scopus (13) Google Scholar, 34Le-Ruppert K. Masters J.R. Knuechel R. Seegers S. Tainsky M.A. Hofstaedter F. Buettner R. Int. J. Cancer. 1992; 51: 646-651Crossref PubMed Scopus (17) Google Scholar), in contrast to F9 cells. Longer duration of treatment with 1 μm retinoic acid for 2 weeks still failed to induce Dab2 expression in PA-1 cells (data not shown). The lack of Dab2 induction occurs at the transcriptional level, because no changes in DAB2 mRNA were observed (Fig. 2 B). RNA from ES2 cells, a Dab2-positive ovarian cancer cell line (5Fazili Z. Sun W. Mittelstaedt S. Cohen C. Xu X.X. Oncogene. 1999; 18: 3104-3113Crossref PubMed Scopus (120) Google Scholar), was used as a positive control. In parallel experiments, retinoic acid inhibited the growth of F9 cells in a time (Fig. 3 A)- and dose-dependent manner (Fig. 3 C) and also caused morphological changes of the cells in culture (Fig. 3 D). Suppression of cell growth correlated with the induction of Dab2 expression since both occurred at day 3 after treatment with retinoic acid. F9 cells treated with retinoic acid for 4 days were well separated and dispersed compared with non-treated cells, which appeared tightly packed and physically connected. In contrast, retinoic acid had no effect on PA-1 cell growth (Fig. 3, B and C), although a morphological change was seen, in agreement with previous reports (19Soprano D.R. Chen L.X. Wu S. Donigan A. Borghaei R.C. Soprano K.J. Oncogene. 1996; 12: 577-584PubMed Google Scholar, 33Taylor D.D. Taylor C.G. Black P.H. Jiang C.G. Chou I.N. Differentiation. 1990; 43: 123-130Crossref PubMed Scopus (13) Google Scholar, 34Le-Ruppert K. Masters J.R. Knuechel R. Seegers S. Tainsky M.A. Hofstaedter F. Buettner R. Int. J. Cancer. 1992; 51: 646-651Crossref PubMed Scopus (17) Google Scholar). In PA-1 cells treated with 1 μmretinoic acid for 4 days (Fig. 3 E), cells appear to be less elongated and the nuclei more pronounced. Thus, retinoic acid induces morphological changes and cell growth suppression in F9 cells and induces morphological changes but no growth suppression in PA-1 cells. Resistance to retinoic acid-induced growth suppression, therefore, correlates with a lack of Dab2 induction. To examine the effect of Dab2 on cell growth and morphology, a Dab2 expression construct was transfected into both F9 and PA-1 cells. In F9 cells transfected with Dab2, only three G418-resistant colonies were selected compared with 64 resistant colonies of vector controls in parallel transfection. After expansion of the three Dab2-transfected clones, none were found to express the Dab2 protein as detected by Western blotting. We then transfected F9 cells with mouse Dab2 p96 construct under the control of the metallothionein promoter (pMT-CB6+ vector). In 48 clones selected for analysis, at least 6 clones appear to express the Dab2 p96 protein (Fig. 4 A). However, we have also observed that the ZnSO4-induced F9 cells undergo differentiation without retinoic acid; the cells also express the p67 form of Dab2 (although these were transfected with the p96 form of Dab2), and the cells also express GATA-4, GATA-6, collagen IV α2, and laminin, which are markers for differentiated endoderm cells. Thus, we conclude that it is not possible to obtain stable Dab2-expressing F9 cells without also inducing spontaneous retinoic acid-independent differentiation. However, we are able to transiently express Dab2 by adenoviral approach (Fig. 4 B) without inducing differentiation of the F9 cells, as judged by the lack of expression of the p67 spliced form of Dab2, GATA-4, and GATA-6. Dab2 expression by the adenoviral approach suppresses F9 cell growth (Fig. 4 C), suggesting that retinoic acid-induced Dab2 expression is responsible for the cell growth inhibitory activity of retinoic acid in F9 cells. There are undoubtedly many differences in the genetic background and properties of mouse F9 and human PA-1 teratocarcinoma cells, although both cell lines have some properties of embryonic stem cells. Although both are undifferentiated and multipotent, PA-1 cells synthesize collagen IV and laminin (32Zeuthen J. Norgaard J.O. Avner P. Fellous M. Wartiovaara J. Vaheri A. Rosen A. Giovanella B.C. Int. J. Cancer. 1980; 25: 19-32Crossref PubMed Scopus (150) Google Scholar, 33Taylor D.D. Taylor C.G. Black P.H. Jiang C.G. Chou I.N. Differentiation. 1990; 43: 123-130Crossref PubMed Scopus (13) Google Scholar, 34Le-Ruppert K. Masters J.R. Knuechel R. Seegers S. Tainsky M.A. Hofstaedter F. Buettner R. Int. J. Cancer. 1992; 51: 646-651Crossref PubMed Scopus (17) Google Scholar), unlike F9 cells, which do not express collagen IV and laminin before retinoic acid-induced differentiation (26Faria T.N. Mendelsohn C. Chambon P. Gudas L.J. J. Biol. Chem. 1999; 274: 26783-26788Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). After transfection of PA-1 cells with a Dab2 expression construct, 16 colonies were selected compared with 54 colonies from vector-transfected controls. All of the Dab2-transfected colonies developed much more slowly (estimated to be 20-fold less based on cell number) than colonies from vector-transfected controls, as shown in Fig. 5 A for a typical example of a G418-selected colony. Under identical culture conditions, the Dab2-transfected cells appeared well separated from each other within a colony, whereas the vector-transfected control cells in a colony were aggregated and indistinguishable from parental cells. In an earlier passage with a cell number of about 1 × 105cells/colony, Dab2 expression was detected. Only the p96 form of Dab2 but not the p67 form was expressed, suggesting that Dab2 expression was the result of cDNA transfection and not spontaneous differentiation. However, as cultures were expanded, the morphological difference diminished, and Dab2 expression was gradually lost in most of the clones. For three colonies, Dab2 expression remained after several passages, and the morphological changes, although not as obvious as for the cells in earlier passages, were still apparent compared with vector-transfected cells (Fig. 5 B). Additionally, these Dab2-expressing cells exhibited a reduced growth rate compared with vector-transfected controls (Fig. 5 C). The ability to form colonies on agar plates was suppressed upon Dab2 expression (Fig. 5 D). Therefore, transfection experiments indicate that expression of Dab2 suppresses cell proliferation and anchorage-independent colony formation and alters cell-cell adhesion. Moreover, these changes correlate well with alterations in the cell cycle. Under identical culture conditions as described above, the two transfected PA-1 clones with detectable Dab2 expression (clones 9 and 13) had an increase in the percentage of cells in G1 and a corresponding decrease of cells in S phase compared with vector-transfected or parental cells (Table I). Thus, Dab2 inhibits cell growth by suppressing G1 phase progression, which is similar to the effect of retinoic acid on the cell cycle (17Wu S. Donigan A. Platsoucas C.D. Jung W. Soprano D.R. Soprano K.J. Exp. Cell Res. 1997; 232: 277-286Crossref PubMed Scopus (53) Google Scholar, 18Faria T.N. LaRosa G.J. Wilen E. Liao J. Gudas L.J. Mol. Cell. Endocrinol. 1998; 143: 155-166Crossref PubMed Scopus (47) Google Scholar, 26Faria T.N. Mendelsohn C. Chambon P. Gudas L.J. J. Biol. Chem. 1999; 274: 26783-26788Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).Table ICell flow cytometry analysisCellG1SG2 + M% total cellsPA-1482725Vector512425Dab2 Clone 975817Dab2 Clone 1366826PA-1 cells were transfected with vector or a human Dab2 expression construct and selected in DMEM containing 10% FBS and zeomycin. Two clones (Clone 9 and 13) were isolated that stably expressed D
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