Krüppel-like Factor 4 (Gut-enriched Krüppel-like Factor) Inhibits Cell Proliferation by Blocking G1/S Progression of the Cell Cycle
2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês
10.1074/jbc.m101194200
ISSN1083-351X
AutoresXinming Chen, David C. Johns, Deborah E. Geiman, Eduardo Marbán, Duyen T. Dang, Gina Hamlin, Ronggai Sun, Vincent W. Yang,
Tópico(s)Genetic Syndromes and Imprinting
ResumoKrüppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G1/S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducibleKLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G1/S boundary of the cell cycle. Krüppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G1/S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducibleKLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G1/S boundary of the cell cycle. Krüppel-like factor 4 adenovirus containing EGFP adenovirus containing EGFP and KLF4 Chinese hamster ovary ecdysone receptor enhanced green fluorescence protein fluorescence activated cell sorting fetal bovine serum lung Krüppel-like factor ponasterone A phosphate-buffered saline retinoid X receptor ecdysone receptor element (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt Krüppel-like factor 4 (KLF4;1 also called gut-enriched Krüppel-like factor) is a recently identified zinc finger-containing transcription factor that is related to theDrosophila melanogaster segmentation gene product, Krüppel (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 2Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 3Schuh R. Aicher W. Gual U. Cote S. Preiss A. Maier D. Seifert E. Nauber U. Shroder C. Kemler R. Jackle H. Cell. 1986; 47: 1025-1032Abstract Full Text PDF PubMed Scopus (349) Google Scholar). It belongs to a rapidly expanding family of mammalian Krüppel-like factors with erythroid Krüppel-like factor (KLF1) (4Miller I.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (667) Google Scholar) serving as a prototype member (5Turner J. Crossley M. Trends Biochem. Sci. 1999; 24: 236-240Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 6Philipsen S. Suske G. Nucleic Acids Res. 1999; 27: 2991-3000Crossref PubMed Scopus (541) Google Scholar, 7Dang D.T. Pevsner J. Yang V.W. Int. J. Biochem. Cell Biol. 2000; 32: 1103-1121Crossref PubMed Scopus (380) Google Scholar). Within this family, KLF4 is most closely related to KLF1 and KLF2 (lung Krüppel-like factor or LKLF) (8Anderson K.P. Kern C.B. Crable S.C. Lingrel J.B. Mol. Cell. Biol. 1995; 15: 5957-5965Crossref PubMed Scopus (231) Google Scholar). Through studies involving gene knockout, all three KLFs have been shown to be important in regulating development of tissue-specific functions (9Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (492) Google Scholar, 10Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (540) Google Scholar, 11Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (349) Google Scholar, 12Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (315) Google Scholar, 13Wani M.A. Means Jr., R.T. Lingrel J.B. Transgenic Res. 1998; 7: 229-238Crossref PubMed Scopus (120) Google Scholar, 14Wani M.A. Wert S.E. Lingrel J.B. J. Biol. Chem. 1999; 274: 21180-21185Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 15Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (659) Google Scholar). KLF4, for example, was shown to be necessary for the development of the barrier function of the skin (15Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (659) Google Scholar). Expression of KLF4 is particularly enriched in epithelial tissues such as the intestine and skin (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 2Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 15Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (659) Google Scholar, 16Ohnishi S. Laub F. Matsumoto N. Asaka M. Ramirez F. Yoshida T. Terada M. Dev. Dyn. 2000; 217: 421-429Crossref PubMed Scopus (77) Google Scholar, 17Jenkins T.D. Opitz O.G. Okano J. Rustgi A.K. J. Biol. Chem. 1998; 273: 10747-10754Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In the intestinal epithelium, KLF4 is abundantly expressed in the post-mitotic, differentiated epithelial cells outside the proliferating zone of crypt cells (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 2Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 16Ohnishi S. Laub F. Matsumoto N. Asaka M. Ramirez F. Yoshida T. Terada M. Dev. Dyn. 2000; 217: 421-429Crossref PubMed Scopus (77) Google Scholar). In addition, in conjunction with several other KLFs, KLF4 is highly expressed in naive, quiescent B lymphocytes and becomes rapidly down-regulated upon mitogenic activation (18Glynne R. Ghandour G. Rayner J. Mack D.H. Goodnow C.C. Immunol. Rev. 2000; 176: 216-246Crossref PubMed Scopus (149) Google Scholar). The in vitro pattern of expression of KLF4 mirrors its in vivoexpression. For example, KLF4 is primarily expressed in a growth-arrested state brought on by serum deprivation, contact inhibition, DNA damage, or growth-inhibitory cytokines (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 19Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Chen Z.Y. Shie J. Tseng C. FEBS Lett. 2000; 477: 67-72Crossref PubMed Scopus (58) Google Scholar). Conversely, expression of KLF4 is decreased in conditions associated with increased proliferation such as in neoplasm of the intestinal tract (21Dang D.T. Bachman K.E. Mahatan C.S. Dang L.H. Giardiello F.M. Yang V.W. FEBS Lett. 2000; 476: 203-207Crossref PubMed Scopus (88) Google Scholar, 22Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-814Crossref PubMed Google Scholar). These studies, therefore, demonstrate a correlation between KLF4 expression and a process of growth arrest. Consistent with the notion that KLF4 may be important in regulating growth, previous studies have demonstrated that KLF4 is an inhibitor of cellular proliferation. For example, constitutive expression ofKLF4 in transfected fibroblasts or colon cancer cells resulted in the inhibition of DNA synthesis (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 22Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-814Crossref PubMed Google Scholar). This may in part be explained by the ability of KLF4 to activate the p21WAF1/Cip1 promoter (19Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), a cell cycle inhibitor, and to suppress the cyclin D1 promoter (23Shie J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar), a positive cell cycle regulator. However, the exact mechanism by which KLF4 inhibits cell proliferation is somewhat unclear, with evidence suggesting that it causes either apoptosis (20Chen Z.Y. Shie J. Tseng C. FEBS Lett. 2000; 477: 67-72Crossref PubMed Scopus (58) Google Scholar) or cell cycle arrest (22Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-814Crossref PubMed Google Scholar). To further define the molecular mechanism by which KLF4 regulates the cell cycle, we developed a system using an inducible promoter to control the expression of KLF4. We show by two independent means that induced KLF4 expression results in an accumulation of cells in the G1 phase of the cell cycle with a concomitant decrease of those in the S phase. The results of our study therefore provide evidence that KLF4 is a cell cycle regulator and does so by creating a block in the G1/S transition phase of the cell cycle. pVgRXR, a plasmid engineered to constitutively express both the VgEcR and RXR receptor subunit, was obtained from Invitrogen (Carlsbad, CA). VgEcR was generated by fusing the DNA-binding domain of the D. melanogaster ecdysone receptor (EcR) to a modified transactivation domain of the herpes simplex virus 1 VP16 protein (24No D. Yao T.P. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3346-3351Crossref PubMed Scopus (758) Google Scholar, 25Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (350) Google Scholar). RXR (retinoid X receptor) is the mammalian homologue of USP (ultraspiracle), the natural partner to theDrosophila ecdysone receptor (26Yao T.P. Forman B.M. Jiang Z. Cherbas L. Chen J.D. McKeown M. Cherbas P. Evans R.M. Nature. 1993; 366: 476-479Crossref PubMed Scopus (794) Google Scholar, 27Yao T.P. Segraves W.A. Oro A.E. McKeown M. Evans R.M. Cell. 1992; 71: 63-72Abstract Full Text PDF PubMed Scopus (628) Google Scholar). Upon binding to ecdysone, the heterodimeric VgEcR and RXR form a functional ecdysone receptor that is required for the optimal interaction with the ecdysone receptor element (EcRE). The plasmid pAdLoxEGI-KLF4 was obtained by subcloning the full-length coding region of the KLF4 cDNA into pAdLoxEGI (28Johns D.C. Marx R. Mains R.E. O'Rourke B. Marban E. J. Neurosci. 1999; 19: 1691-1697Crossref PubMed Google Scholar). The latter was constructed from the pAdLox plasmid (29Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar) by substituting the ecdysone inducible promoter from pIND (Invitrogen) for the cytomegalovirus promoter and by inserting an expression cassette containing the enhanced green fluorescence protein (EGFP) (30Zhang G. Gurtu V. Kain S.R. Biochem. Biophys. Res. Commun. 1996; 227: 707-711Crossref PubMed Scopus (325) Google Scholar), which was followed by an internal ribosome entry site (31Johns D.C. Nuss H.B. Marban E. J. Biol. Chem. 1997; 272: 31598-31603Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). TheKLF4 cDNA was inserted between the XhoI andEcoRI sites in the multiple cloning sites following the internal ribosome entry site. Transfection was accomplished using the LipofectAMINE reagent from Life Technologies (Gaithersberg, MD). To test the co-expression of EGFP andKLF4, EcR-CHO cells, a Chinese hamster ovary (CHO) cell line containing the stably integrated pVgRXR vector, were cultured on glass coverslips in 6-well dishes. Cells were transfected with pAdLoxEGI-KLF4 and treated for 24 h with 5 µm ponasterone A (Invitrogen). The coverslip was washed twice with phosphate-buffered saline (PBS) and fixed in 3.5% paraformaldehyde in PBS for 8 min followed by washing twice with PBS. Cells were then permeabilized in 0.1% Nonidet P-40 in PBS for 15 min, washed twice with PBS, and incubated in 10% fetal bovine serum (FBS) in PBS for 20 min. Fifty µl of a 1:100 diluted anti-KLF4 serum in PBS containing 10% FBS was added to the coverslip and incubated at 4 °C overnight. The coverslip was washed 3 times with 10% FBS in PBS followed by the addition of 100 µl of a 1:200 dilution of Cy3-labeled secondary antibody and incubated for 1 h at room temperature. The coverslip was then washed with 0.01% Tween 20 in PBS for 30 min and placed face down in 100 µl of mounting media containing 50% glycerol in PBS. Stained cells were visualized under a confocal microscope using filters designed to detect GFP and Cy3. The primary antibody was eliminated in control experiments. The EcR-RKO/pAdLoxEGI-KLF4 cell line was generated by co-transfecting pVgRXR and pAdLoxEGI-KLF4 at a molar ratio of 1:20 into the RKO human colon cancer cells. Two days following transfection, 150 µg/ml Zeocin was added to the medium to select for resistant clones. To identify clones that contained the inducible KLF4, cells were treated with 5 µm ponasterone A for 24 h. Individual clones that exhibited green fluorescence were expanded and further sorted by Star Plus (Becton Dickinson) following induction to enrich for cells stably expressing EGFP. The level of KLF4induction was tested by Northern and Western blot analyses upon hormonal treatment. EcR-RKO cells were similarly obtained as above except that transfection was performed with pVgRXR alone. Zeocin-resistant clones were selected and the degree of RXR expression measured by Western blot analysis. The recombinant adenovirus containing EGFP and KLF4 (AdEGI-KLF4) or GFP alone (AdEGI) was generated by Cre-lox recombination of purified ψ5 viral DNA (29Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar) and pAdLoxEGI-KLF4 or pAdLoxEGI, respectively, as previously described (28Johns D.C. Marx R. Mains R.E. O'Rourke B. Marban E. J. Neurosci. 1999; 19: 1691-1697Crossref PubMed Google Scholar, 29Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar). The recombinant products were plaque-purified and expanded by infecting HEK293 cells for 8 h. Forty-eight hours after infection, cells were freeze-thawed 3 cycles to release the viruses, which were purified by CsCl gradient centrifugation to achieve a titer of ∼1010 plaque forming units/ml. EcR-RKO cells were grown to 90% confluence in 10-cm dishes and replenished with fresh media containing 2% FBS followed by the addition of 108 plaque forming units of recombinant virus per dish. Infected cells were incubated at 37 °C for 6–8 h, at which time the media were changed and incubation continued overnight. Cells were then treated with 5 µm ponasterone A for 24 h and then collected for further analyses. RNA was extracted from cells using the TriZol reagent (Life Technologies) following the manufacturers recommendations. Twenty µg of total RNA was resolved by electrophoresis in 1.2% agarose gels containing 2.4 mformaldehyde and transblotted onto nylon membranes (Hybond-N; Amersham Pharmacia Biotech). Hybridization and washing were performed under high stringency conditions using radioactively labeled KLF4 and glyceraldehyde-3-phosphate dehydrogenase cDNA probes. Western blot analyses were performed using standard procedures. Protein samples were dissolved in a loading buffer (60 mm Tris-HCl, pH 6.8, 2% SDS, 100 mm dithiothreitol, and 0.01% bromphenol blue), heated at 100 °C for 3 min, and loaded onto a SDS-polyacrylamide gel in running buffer containing 25 mmTris-HCl, pH 8.3, 250 mm glycine, and 0.1% SDS. At the completion of electrophoresis, proteins were electrophoretically transferred to a membrane, which was immunoblotted with a rabbit anti-KLF4 serum (1:1,000 dilution; Ref. 1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar) or anti-RXR serum (1:1,000 dilution; Santa Cruz). Following incubation with the secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit IgG; Santa Cruz), KLF4 or RXR was visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech). EcR-RKO/pAdLoxEGI-KLF4 cells were plated on 60-mm dishes at a density of 2.8 × 105cells/dish. After 24 h, ponasterone A was added to a final concentration of 5 µm. Media containing ponasterone A were changed every other day. Control cells received vehicle (ethanol) only. Cell numbers were measured with a hemocytometer daily up to 8 days following seeding. MTS assays were performed based using the manufacturers recommendations (Promega, Madison, WI). EcR-RKO and EcR-RKO/pAdLoxEGI-KLF4 cells were plated in 96-well microtiter plates at a density of 1.0 × 103 cells/well in Dulbecco's modified Eagle's medium with 10% FBS and 150 µg/ml Zeocin. After 24 h, the medium was changed to Dulbecco's modified Eagle's medium containing 10% FBS, 150 µg/ml zeocin, and either 5 µm ponasterone A or vehicle alone. The number of cells at this time (t = day 0) and at 1, 3, and 5 days was determined using the colorimetric MTS assay. Results are depicted as absorbance at 490 nm as a function of time. DNA content was measured following staining of cells with propidium iodide. EcR-RKO/pAdLoxEGI-KLF4 cells were treated with 5 µm ponasterone A or vehicle alone for 24 h. Cells were subsequently trypsinized, washed once in cold PBS, and fixed in 70% ethanol at −20 °C overnight. Fixed cells were pelleted and stained in the propidium iodide solution (50 µg/ml propidium iodide, 50 µg/ml RNase A, 0.1% Triton X-100, and 0.1 mm EDTA) in the dark at 4 °C for 1 h prior to flow cytometric quantification of DNA by a FACScan (Becton Dickinson). Similarly, FACS analyses were conducted in EcR-RKO cells infected with recombinant AdLoxEGI-KLF4 or AdLoxEGI viruses followed by treatment with 5 µm ponasterone A or vehicle alone for 24 h. To investigate the mechanism by which KLF4 regulates the cell cycle, we first established an inducible system in whichKLF4 expression could be controlled. We generated a DNA construct called pAdLoxEGI-KLF4 that would produce a bicistronic message of enhanced green fluorescence protein (EGFP) and KLF4 separated by an internal ribosomal entry site (31Johns D.C. Nuss H.B. Marban E. J. Biol. Chem. 1997; 272: 31598-31603Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Expression ofEGFP and KLF4 was controlled by a promoter that contained a response element to a hormone-activated EcR (24No D. Yao T.P. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3346-3351Crossref PubMed Scopus (758) Google Scholar). A CHO cell line, called EcR-CHO, that had a stably integrated dimeric ecdysone receptor and RXR (26Yao T.P. Forman B.M. Jiang Z. Cherbas L. Chen J.D. McKeown M. Cherbas P. Evans R.M. Nature. 1993; 366: 476-479Crossref PubMed Scopus (794) Google Scholar, 27Yao T.P. Segraves W.A. Oro A.E. McKeown M. Evans R.M. Cell. 1992; 71: 63-72Abstract Full Text PDF PubMed Scopus (628) Google Scholar) derived from the plasmid pVgRXR was transiently transfected with pAdLoxEGI-KLF4 and treated with an ecdysone analogue, ponasterone A. As seen in Fig.1, GFP and KLF4 were produced in the same transfected cells (panels A and B, respectively), as evidenced by direct fluorescence visualization of the former and by immunocytochemical staining of the latter. As a control, transfected cells (Fig. 1 C) from which the primary KLF4 antibody was eliminated during staining lacked any fluorescence (Fig.1 D). These results indicate that the pAdLoxEGI-KLF4 construct was able to produce both proteins in transfected cells upon induction. We then established a cell line that contained stably integrated pVgRXR and pAdLoxEGI-KLF4 by co-transfecting a human colon cancer cell line, RKO. Clonal derivatives were selected by the antibiotic Zeocin. We chose RKO cells because of their exceedingly low level ofKLF4 mRNA, which is probably due to mutations in upstream genes that regulate KLF4 expression (32Dang, D. T., Mahatan, C. S., Dang, L. H., Agboola, I. A., and Yang, V. W. (2001) Oncogene, in press.Google Scholar). A stably transfected clone, called EcR-RKO/pAdLoxEGI-KLF4, was treated with ponasterone A or vehicle alone for 24 h and visualized with a fluorescence microscope. As shown in Fig.2 A, the addition of ponasterone A resulted in the appearance of numerous green cells in the culture (+PA), whereas, the uninduced control cells exhibited no fluorescence (−PA). The addition of ponasterone A also resulted in a significant increase in the level of the KLF4 transcripts as demonstrated by Northern blot analysis (Fig. 2 B). To begin investigating the effect of induced KLF4 expression on cell proliferation, we seeded EcR-RKO/pAdLoxEGI-KLF4 cells in low densities and treated them with ponasterone A or vehicle alone for up to 8 days. Cell numbers were assessed by direct daily counting (Fig.3 A) or by MTS assay every other day (Fig. 3 B). As seen, the ponasterone A-treated cells did not proliferate during the first 4 days of treatment when compared with the control cells. After 4 days of treatment, the number of induced cells did begin to increase but at a slower rate compared with the uninduced cells. As demonstrated by the Western blot analysis in Fig. 3 C, the level of KLF4 protein was low in uniduced cells (day 0) but was increased 1 day following induction and maintained at a similar level up to 6 days. These results suggest that the decreased proliferation in induced cells is a consequence of KLF4 production. To address the mechanism by which KLF4 inhibits cell proliferation, we performed FACS analyses of EcR-RKO/pAdLoxEGI-KLF4 cells treated or not with ponasterone A for 24 or 48 h. As shown in Fig.4 A, a great majority of cells exhibited green fluorescent after 24 h of ponasterone A treatment. This was accompanied by a change in the cell cycle profile as illustrated in Fig. 4 B. Specifically, the induction ofKLF4 caused a statistically significant increase in the percentage of cells in the G1 phase of the cell cycle and a statistically significant decrease in the percentage of cells in the S phase (Fig. 4 C). The difference in the percentage of cells in the G2 phase between treated and control groups was not significant. In addition, FACS analyses showed no evidence of apoptosis in the induced cells, which would have been manifested by a DNA content, that is less than G1 as in Fig. 4 B. The results obtained after 48 h of induction were similar to those at 24 h (results not shown). Importantly, accompanying the G1/S block associated with hormonal induction was a concomitant increase in the amount of p21WAF1/Cip1 mRNA (Fig. 4 C, inset). These results suggest that the ability of KLF4 in blocking the cell cycle is mediated by p21WAF1/Cip1. As an alternative and independent means to measure the effect of KLF4 on the cell cycle, we constructed a recombinant adenovirus, called AdEGI-KLF4, using the pAdLoxEGI-KLF4 plasmid as a shuttle vector (see “Experimental Procedures”). We also established a RKO cell line, called EcR-RKO, which had a stably integrated pVgRXR construct (Fig.5 A, lane 3). Following infection with the AdEGI-KLF4 recombinant virus, EcR-RKO cells were treated with ponasterone A or vehicle alone for 24 h and the degree of KLF4 expression assessed by Northern and Western blot analyses. As shown in Fig. 5 B, both the levels of KLF4 mRNA and protein were significantly increased in infected and induced cells when compared with infected but uninduced cells (Fig.5 B, upper and lower panel, respectively). The induced KLF4 expression was accompanied by the appearance of green fluorescence in cells treated with ponasterone A (Fig.5 C). To control for a possible effect of EGFP on the cell cycle, we also generated a recombinant adenovirus, called AdEGI that contained only EGFP. As seen in Fig. 5 D, EcR-RKO cells infected with the AdEGI virus and induced with ponasterone A also exhibited intense green fluorescence at 24 h. Importantly, FACS analysis of infected cells showed only those infected by AdEGI-KLF4 and induced with ponasterone A had a statistically significant increase in the G1 population and a statistically significant decrease in the S population when compared with the infected but uninduced cells (Fig. 5 E). There was no difference in the cell cycle profiles of AdEGI virus-infected cells between induced and uninduced conditions (Fig. 5 F). These results suggest that the alteration in cell cycle profile upon ponasterone A addition is the result of induction in KLF4 and not in EGFPexpression. KLF4 was first identified some 5 years ago as a gene whose expression is temporally associated with growth arrest (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). This correlation has been documented in several independent in vitro conditions (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 19Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Chen Z.Y. Shie J. Tseng C. FEBS Lett. 2000; 477: 67-72Crossref PubMed Scopus (58) Google Scholar, 23Shie J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar). In vivo, the predominant expression of KLF4 in differentiated cells of several epithelial tissues also suggests that it is a growth arrest-specific gene (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 2Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 15Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (659) Google Scholar, 16Ohnishi S. Laub F. Matsumoto N. Asaka M. Ramirez F. Yoshida T. Terada M. Dev. Dyn. 2000; 217: 421-429Crossref PubMed Scopus (77) Google Scholar, 33Panigada M. Porcellini S. Sutti F. Doneda L. Pozzoli O. Consalez G.G. Guttinger M. Grassi F. Mech. Dev. 1999; 81: 103-113Crossref PubMed Scopus (46) Google Scholar). It is particularly telling that KLF4, along with several other Krüppel-like factor-encoding genes, is highly expressed in naive, quiescent B lymphocytes and is rapidly down-regulated upon activation due to mitogenic stimulation (18Glynne R. Ghandour G. Rayner J. Mack D.H. Goodnow C.C. Immunol. Rev. 2000; 176: 216-246Crossref PubMed Scopus (149) Google Scholar). These observations suggest that KLF4 may have an inhibitory function in regulating cell proliferation. Consistent with this notion, KLF4 was shown to inhibit DNA synthesis when constitutively produced in transfected cells (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). The present study established the mechanism by which KLF4 exerts its negative effect on cell growth. This was accomplished by the development of an inducible system for KLF4 expression. By either stable transfection or adenovirus-mediated infection of RKO cells, we demonstrated that induced expression of KLF4caused a significantly decreased rate of proliferation, which was likely due to a block in the cell cycle at the G1/S boundary. The result of our study is consistent with that from a previous one, which examined the effect of constitutive KLF4expression in a stably transfected colon cancer cell line, HT-29 (22Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-814Crossref PubMed Google Scholar). It is also consistent with the finding that, among the genes regulated by KLF4 are p21WAF1/Cip1 and cyclin D1, two genes with key, albeit opposite, effects in controlling G1/S transition in the cell cycle (35Donjerkovic D. Scott D.W. Cell Res. 2000; 10: 1-16Crossref PubMed Scopus (129) Google Scholar, 36Harper J.W. Cancer Surv. 1997; 29: 91-107PubMed Google Scholar, 37Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2623) Google Scholar). As KLF4 is an activator of transcription of p21WAF1/Cip1 (19Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) and a suppressor of transcription of cyclin D1 (23Shie J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar), a logical consequence of KLF4s activation is the impediment in the transition from G1 to S phase as observed in the present study. The physiological implication of our finding extends our previous observation that cell cycle arrest due to DNA damage is accompanied by p53-dependent activation of KLF4 expression (19Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Moreover, an activated KLF4 transcriptionally induces p21WAF1/Cip1 expression. This is further supported by the present study showing that the level of p21WAF1/Cip1 transcript is significantly increased due to the induction of KLF4(Fig. 4 C). Combining the results of these studies, it appears likely that KLF4 is an essential member of the p53 tumor suppressor network of cell cycle regulators. A recent study showed that expression of KLF4 is up-regulated by treatment of the human colon cancer cell line, HT-29, with the cytokine interferon-γ (20Chen Z.Y. Shie J. Tseng C. FEBS Lett. 2000; 477: 67-72Crossref PubMed Scopus (58) Google Scholar). In this system, interferon-γ induced growth arrest in HT-29 cells by causing apoptosis. Moreover, transient transfection of several colon cancer cell lines with aKLF4-expressing plasmid was shown to promote apoptosis as evidenced by DNA fragmentation (20Chen Z.Y. Shie J. Tseng C. FEBS Lett. 2000; 477: 67-72Crossref PubMed Scopus (58) Google Scholar). It was proposed thatKLF4 may be a downstream target gene of interferon-γ and may mediate the apoptotic effect of interferon-γ. In our system, however, we did not observe any evidence of apoptosis by FACS or DNA staining (results not shown) during the first 48 h of induction in either stably transfected or infected cells. As the same group has reported in a different study that stable expression of KLF4in HT-29 cells caused cell cycle arrest but not apoptosis (22Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-814Crossref PubMed Google Scholar), it is unclear at this time whether KLF4 is in fact a pro-apoptotic factor. The negative effect of KLF4 on the cell cycle progression suggests that KLF4 may play an important role in regulating the transition of cells from a proliferative to a non-proliferative state such as those observed in vitro and in vivo (1Shields J.M. Christy R.J. Yang V.W. J. Biol. Chem. 1996; 271: 20009-20017Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 2Garrett-Sinha L.A. Eberspaecher H. Seldin M.F. de Crombrugghe B. J. Biol. Chem. 1996; 271: 31384-31390Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 15Segre J.A. Bauer C. Fuchs E. Nat. Genet. 1999; 22: 356-360Crossref PubMed Scopus (659) Google Scholar, 16Ohnishi S. Laub F. Matsumoto N. Asaka M. Ramirez F. Yoshida T. Terada M. Dev. Dyn. 2000; 217: 421-429Crossref PubMed Scopus (77) Google Scholar,18Glynne R. Ghandour G. Rayner J. Mack D.H. Goodnow C.C. Immunol. Rev. 2000; 176: 216-246Crossref PubMed Scopus (149) Google Scholar, 19Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Chen Z.Y. Shie J. Tseng C. FEBS Lett. 2000; 477: 67-72Crossref PubMed Scopus (58) Google Scholar, 23Shie J.L. Chen Z.Y. Fu M. Pestell R.G. Tseng C.C. Nucleic Acids Res. 2000; 28: 2969-2976Crossref PubMed Google Scholar, 33Panigada M. Porcellini S. Sutti F. Doneda L. Pozzoli O. Consalez G.G. Guttinger M. Grassi F. Mech. Dev. 1999; 81: 103-113Crossref PubMed Scopus (46) Google Scholar). It may also be reasonable to speculate that one of KLF4s functions in terminally differentiated epithelial cells in organs such as the gut or skin is to prevent the cells from re-entering the cell cycle. Conversely, in situations where there is increased proliferation such as that encountered in neoplasm, one may expect to see a decrease in KLF4 expression, which may in turn contribute to the tumorigenic phenotype. Indeed, expression ofKLF4 is diminished in benign intestinal adenomas when compared with the surrounding normal intestinal mucosa in both mouse and human models of a hereditary polyposis syndrome (21Dang D.T. Bachman K.E. Mahatan C.S. Dang L.H. Giardiello F.M. Yang V.W. FEBS Lett. 2000; 476: 203-207Crossref PubMed Scopus (88) Google Scholar). Similarly, levels of KLF4 mRNA have been shown to be lower in sporadic cases of colonic adenomas and carcinomas (22Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-814Crossref PubMed Google Scholar). However, it should be cautioned that the expression pattern of KLF4 in cancer tissues might not always be consistent with its putative role as a tumor suppressor. For example, increased expression ofKLF4 has been observed in dysplastic squamous epithelium and squamous cell carcinoma of the oral cavity (38Foster K.W. Ren S. Louro I.D. Lobo-Ruppert S.M. McKie-Bell P. Grizzle W. Hayes M.R. Broker T.R. Chow L.T. Ruppert J.M. Cell Growth & Differ. 1999; 10: 423-434PubMed Google Scholar), and in breast cancer (39Foster K.W. Frost A.R. McKie-Bell P. Lin C.Y. Engler J.A. Grizzle W.E. Ruppert J.M. Cancer Res. 2000; 60: 6488-6495PubMed Google Scholar). Moreover, evidence suggests that KLF4 may be deregulated or misexpressed in certain tumorous tissues (22Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-814Crossref PubMed Google Scholar, 38Foster K.W. Ren S. Louro I.D. Lobo-Ruppert S.M. McKie-Bell P. Grizzle W. Hayes M.R. Broker T.R. Chow L.T. Ruppert J.M. Cell Growth & Differ. 1999; 10: 423-434PubMed Google Scholar). A potential explanation for the apparent discrepancies stated above is that certain tumors may contain mutated forms of KLF4, which may exert a dominant negative effect on cell function. Previous studies indicating that KLF4 interacts with multiple regulatory proteins including Sp1 (40Zhang W. Shields J.M. Sogawa K. Fujii-Kuriyama Y. Yang V.W. J. Biol. Chem. 1998; 273: 17917-17925Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), p53 (19Zhang W. Geiman D.E. Shields J.M. Dang D.T. Mahatan C.S. Kaestner K.H. Biggs J.R. Kraft A.S. Yang V.W. J. Biol. Chem. 2000; 275: 18391-18398Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), Zf9 (41Okano J. Opitz O.G. Nakagawa H. Jenkins T.D. Friedman S.L. Rustgi A.K. FEBS Lett. 2000; 473: 95-100Crossref PubMed Scopus (59) Google Scholar), and p300/CBP (34Geiman D.E. Ton-That H. Johnson J.M. Yang V.W. Nucleic Acids Res. 2000; 28: 1106-1113Crossref PubMed Google Scholar) distinctly suggest that this latter hypothesis may be a possibility.
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