p21Cip1 and p27Kip1 Induce Distinct Cell Cycle Effects and Differentiation Programs in Myeloid Leukemia Cells

Abstract

The cyclin-dependent kinase (Cdk) inhibitors p21Cip1 and p27Kip1 have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21Cip1 and p27Kip1 in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21Cip1 or p27Kip1 arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21Cip1 arrested cells in both G1 and G2 cell cycle phases, p27Kip1 blocked the G1/S-phase transition. Furthermore, although both p21Cip1 and p27Kip1 associated with Cdk6, only p27Kip1 significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21Cip1 triggered megakaryocytic maturation, whereas p27Kip1 resulted in the expression of erythroid markers. Consistently, p21Cip1 and p27Kip1 were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21Cip1 and p27Kip1 in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable. The cyclin-dependent kinase (Cdk) inhibitors p21Cip1 and p27Kip1 have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21Cip1 and p27Kip1 in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21Cip1 or p27Kip1 arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21Cip1 arrested cells in both G1 and G2 cell cycle phases, p27Kip1 blocked the G1/S-phase transition. Furthermore, although both p21Cip1 and p27Kip1 associated with Cdk6, only p27Kip1 significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21Cip1 triggered megakaryocytic maturation, whereas p27Kip1 resulted in the expression of erythroid markers. Consistently, p21Cip1 and p27Kip1 were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21Cip1 and p27Kip1 in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable. p21Cip1 (hereafter termed p21) and p27Kip1 (hereafter termed p27) belong to the Cip/Kip family of cyclin-dependent kinase (Cdk) 1The abbreviations used are: Cdk, cyclin-dependent kinase; ARA-C, 1-β-arabinofuranosylcytosine; BrdUrd, bromodeoxyuridine; CKI, Cdk inhibitory protein; DAPI, 4′-6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; Rb, retinoblastoma protein. inhibitors (CKIs) and have been extensively characterized as negative regulators of the G1-phase cell cycle progression. p21 and p27 are found in complexes composed of Cdks and their cyclin-activating partners and inhibit their catalytic activities. These interactions are mediated by the conserved amino-terminal portions of the proteins, which enable them to bind both the cyclin and Cdk subunits. Cip/Kip proteins can bind and inhibit a broad spectrum of Cdks, with the strongest inhibitory activity manifested against Cdk2 complexes (1Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5145) Google Scholar, 2Obaya A.J. Sedivy J.M. Cell. Mol. Life Sci. 2002; 59: 126-142Crossref PubMed Scopus (338) Google Scholar). Most intriguingly, Cip/Kip CKIs have also been reported as constituents of active cyclin D-Cdk4/6 complexes, perhaps promoting their assembly (3Soos T.J. Kiyokawa H. Yan J.S. Rubin M.S. Giordano A. DeBlasio A. Bottega S. Wong B. Mendelsohn J. Koff A. Cell Growth & Differ. 1996; 7: 135-146PubMed Google Scholar, 4Blain S.W. Montalvo E. Massague J. J. Biol. Chem. 1997; 272: 25863-25872Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 5LaBaer J. Garrett M.D. Stevenson L.F. 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Several lines of evidence suggest that p21 and p27 exert similar effects on cyclin-Cdk complexes and cell cycle progression as follows: (a) both proteins have conserved sequences in their inhibitory domains; (b) both proteins mediate the inhibition of Cdk2 complexes by a wide range of antimitogenic signals, and (c) the stability of cyclin D-Cdk complexes depends redundantly upon p21 and p27 (6Cheng M. Olivier P. Diehl J.A. Fero M. Roussel M.F. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: 1571-1583Crossref PubMed Scopus (974) Google Scholar). However, other observations indicate that p21 and p27 also have nonoverlapping functions and are not biologically equivalent. (i) p21 and p27 are often differentially expressed; for example, in fibroblasts p27 levels are high in quiescent cells, and p27 destruction is required for the G1/S traverser, whereas p21 levels start low and typically increase during cell cycle entry (1Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5145) Google Scholar). (ii) The efficiency of Cdk4 inhibition in vitro has been reported to be different for p21 and p27 (4Blain S.W. Montalvo E. Massague J. J. Biol. Chem. 1997; 272: 25863-25872Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). (iii) p21 and p27 knock-out mice display different phenotypes (9Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1948) Google Scholar, 10Fero M.L. Rivkin M. Tasch M. Porter P. Carow C.E. Firpo E. Polyak K. Tsai L.H. Broudy V. Perlmutter R.M. Kaushansky K. Roberts J.M. Cell. 1996; 85: 733-744Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar, 11Martin-Caballero J. Flores J.M. Garcia-Palencia P. Collado M. Serrano M. Oncogene. 2004; 23: 8231-8237Crossref PubMed Scopus (31) Google Scholar). Most of the available information on the activities of p21 and p27 concerns their role on cell cycle control and using fibroblasts as the predominant model system. However, p21 and p27 have also been implicated in multiple biological functions unrelated to cell cycle, such as apoptosis, cell motility, and differentiation (12Denicourt C. Dowdy S.F. Genes Dev. 2004; 18: 851-855Crossref PubMed Scopus (157) Google Scholar, 13Coqueret O. Trends Cell Biol. 2003; 13: 65-70Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar). During the hematopoietic differentiation program, both CKIs are up-regulated by multiple agents that trigger myeloid, megakaryocytic, and erythroid lineages (reviewed in Refs. 14Muñoz-Alonso M.J. León J. Boonstra J. G1 Phase Progression. Eurekah Landes Bioscience, Georgetown, TX2003: 236-264Google Scholar and 15Steinman R.A. Oncogene. 2002; 21: 3403-3413Crossref PubMed Scopus (91) Google Scholar). Overexpression of either p21 or p27 in cell lines induces monocytic (16Liu M. Lee M.H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar, 17Rots N.Y. Iavarone A. Bromleigh V. Freedman L.P. Blood. 1999; 93: 2721-2729Crossref PubMed Google Scholar, 18Zhou P. Yao Y. Soh J.W. Weinstein I.B. Anticancer Res. 1999; 19: 4935-4945PubMed Google Scholar, 19Muto A. Kizaki M. Yamato K. Kawai Y. Kamata-Matsushita M. Ueno H. Ohguchi M. Nishihara T. Koeffler H.P. Ikeda Y. Blood. 1999; 93: 2225-2233Crossref PubMed Google Scholar) or megakaryocytic differentiation (20Kikuchi J. Furukawa Y. Iwase S. Terui Y. Nakamura M. Kitagawa S. Kitagawa M. Komatsu N. Miura Y. Blood. 1997; 89: 3980-3990Crossref PubMed Google Scholar, 21Matsumura I. Ishikawa J. Nakajima K. Oritani K. Tomiyama Y. Miyagawa J. Kato T. Miyazaki H. Matsuzawa Y. Kanakura Y. Mol. Cell. 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Biol. 1999; 19: PubMed Scopus Google Scholar). were with of the and and on kinase activity using for Cdk2 or for Cdk6, and cyclin The kinase of and of or of The were for The proteins were by to and using a The were used for p27 and p21 of protein with of or and the on protein were in for and with PBS, for were with a of or FITC, used as the were with to and a of p21 and to the effects of ectopic expression of p21 and p27 on cell cycle progression and differentiation of K562 cells, we with expression of and a K562 cells were with the Several p21 protein expression p21 levels were not to of p21 by in K562 cells E. M.D. P. C. M. M. J. Oncogene. 2000; 19: PubMed Scopus (59) Google Scholar). and the of p21 of which for of the a rapid increase of p21 and protein between and and to The in p21 expression is of activity of in the of and has been for in K562 M.D. A. M. J. Oncogene. 1995; 10: Google Scholar, M. M.D. A. C. J. 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Trends Cell Biol. 2003; 13: 65-70Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar), we performed of in of p21 in were performed with conditionally expressed p27 of p27 expression in is in p27 levels of and to levels of p27 protein and were p27 by in cells in the of for p27 be in the of the protein to the of p21 or p27 to Growth of or K562 cells not to not their In of or cells with resulted in inhibition of results were in other and not p21 not by the of of in the effects of p27 were not were with and with and to the active and cell cycle respectively. in control cells in the or of However, of cells a in the and of cells in the also a G1 In of cells were also cells a in in to cells, of cells in of p21 p27 apoptosis, as by the of a or by of cells not p21 and p27 on gain into the mechanisms by which these CKIs cell cycle we the activity of the G1 Cdk2 activity and inhibited in cells The of activity not by Cdk2 protein expression that the kinase inhibited by p21 p21 in Cdk2 also the of p21 on Cdk4 and and on the kinase activity of cyclin Cdk4 activity not significantly of p21 kinase activities with Cdk4 levels in the p21 protein in Cdk4 both p21 protein also in on activity a of the kinase activity of and to the levels of each The results to the of inhibition of by p21 and that Cdk2 is inhibited In cyclin only a in kinase activity of p21 not the Cdk4 or protein levels To that p21 in these kinase complexes, we p21 and found that cyclin and were kinase activities levels of of the proteins were in control cells in the or of we in associated with p21 with a to of as as and when the of protein found in its of with the of p21 performed with cells, we also found a of Cdk2 kinase activity of p27 and p27 in Cdk2 complexes and Cdk2 protein levels were also p27 can Cdk2 activity a inhibitory as as by its expression of p27 also a inhibition of Cdk6, and cyclin kinase activities The of the Cdk activities that the Cdk2 and Cdk4 inhibition by p27 with that of p21 and and in to p21 p27 also inhibited p27 not Cdk4 and protein levels Furthermore, of p27 Cdk6, and cyclin proteins of p27 the of in its and with the inhibition of Cdk kinase activities these suggest that expression of p21 the cell cycle of K562 cells by Cdk2 and to a activity. However, p27 the cell cycle by not only Cdk2 and Cdk4 also possible for the differences in the inhibition of Cdk activities be that p21 and p27 were to different p21 and p27 were with that have different for their proteins, we the against of p21 and p27 we by the levels of p21 and p27 expressed in and cells with with of proteins, we found that their of expression both CKIs were expressed similar levels in the two cells lines the that the in of to p21 p27 inhibition in a to with the two However, when we the of p21 and p27 with to the we that of p21 and p27 were to to inhibition by p21 by of p21 whereas p27 the in cell cycle progression by p21 or p27 by differentiation. of cells with resulted in with differentiation in cells, as in of cells highly of were in or K562 cells is unique to hematopoietic cells, we cells by and we found a with of in the of a of cells between the and that p21 in these cells cells and to the not of expression of we found that a of megakaryocytic differentiation J. Biol. Chem. Full Text PDF PubMed Google Scholar), up-regulated In two other and R.A. 19: PubMed Scopus Google Scholar), were expressed upon of p21 in the expression of these were in cells not a to p21 is for a to also a of not In a of cells with of erythroid differentiation also of in These cells of erythroid differentiation differentiation and were also a low in control K562 and cells that expression of p21 induces megakaryocytic differentiation in K562 of p27 not of megakaryocytic differentiation or the expression of megakaryocytic not In we a of cells with of erythroid differentiation and of cells a of cells of a high which by a of a to control cells In with the of p27 on cell cycle erythroid differentiation also cells not only also erythroid when the not that expression of p27 erythroid differentiation not megakaryocytic in K562 The K562 cell can be differentiated into the erythroid by with S. Exp. Google Scholar) and into the megakaryocytic by A. P. M. M.D. M. C. C. E. P. N. S. J. Cell Growth & Differ. 1999; 10: Google Scholar). Most we found that in to these p21 and p27 proteins were differentially up-regulated p21 in cells toward megakaryocytic and p27 up-regulated in cells along the erythroid The differentiation in K562 by and were in by cell and expression not of and protein expression that p21 and p27 were by the on p27 protein levels differentiation are in with the expression of p27 of the regulation of in other S. K. Res. PubMed Scopus Google Scholar). The also that p27 not p21 protein in high K562 cells the expression of p21 and p27 during the of K562 cells is in with the differentiation by the ectopic of the p21 of p27 results indicate that p21 is to trigger a megakaryocytic differentiation its in erythroid differentiation is p21 also resulted in a of cells with erythroid has been reported that expressed p21 can p27 R.A. Y. B. C. Oncogene. PubMed Scopus Google Scholar), we a of p27 in to a low of erythroid differentiation. we found that a of p27 protein levels in cells results were also in other not In the of p27 in cells not the expression of To the functional of p27 in cells, we the effects of of p27 by antisense cells a p27 antisense to by p21 to the as cells the of p27 the expression of p27 protein with a of cells to erythroid as with the erythroid of and However, the of megakaryocytic such as or of not by p27 These that p27 is for the low of erythroid differentiation by of that p21 and p27 distinct in p21 the megakaryocytic and p27 in the erythroid p21 and p27 have been reported to redundant in differentiation of myeloid cell lines M.J. León J. Boonstra J. G1 Phase Progression. Eurekah Landes Bioscience, Georgetown, TX2003: 236-264Google Scholar, 15Steinman R.A. Oncogene. 2002; 21: 3403-3413Crossref PubMed Scopus (91) Google Scholar). However, these studies cell lines the differentiation along a single lineage, thus functions of p21 or p27 a multipotent cell differentiation in which we have control of ectopic p21 and p27 expression in the of differentiation Most importantly, the expression levels of p21 and p27 upon were results in model demonstrate distinct of these p21 megakaryocytic and p27 induces erythroid of These effects are in have found that p21 megakaryocytic in the of K562 cells, in with results using other cell myeloid lines (20Kikuchi J. Furukawa Y. Iwase S. Terui Y. Nakamura M. Kitagawa S. Kitagawa M. Komatsu N. Miura Y. Blood. 1997; 89: 3980-3990Crossref PubMed Google Scholar, 21Matsumura I. Ishikawa J. Nakajima K. Oritani K. Tomiyama Y. Miyagawa J. Kato T. Miyazaki H. Matsuzawa Y. Kanakura Y. Mol. Cell. Biol. 1997; 17: 2933-2943Crossref PubMed Scopus (172) Google Scholar). Most p21 protein expression only transiently in which is with of p21 prior to differentiation in myeloid cells B. S. T.J. R.A. 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Blood. 1999; 93: PubMed Google Scholar). further between p21 and p27 in K562 cells is their effects on cell cycle progression. found that both p21 and p27 blocked G1 progression in G1 is the of the inhibition of Cdk2 activity and the of phosphorylation. However, p27 p21 in the G1 and the inhibition of activity to In to expression of p21 also G2 The of p21 in the regulation of the has been in V. Mol. Cell. Biol. 1998; 18: PubMed Google Scholar, A. C. T. S. Sedivy J.M. B. 1998; PubMed Scopus Google Scholar, M. C. Mol. Cell. Biol. 1998; 18: PubMed Google Scholar). Most of of cells, p21 expression to low and which a of the cells not in cells when p27 by of is that of is for the that cells arrested in G2 by p21 expression the to a cell cycle progression. proteins in control and the of in the cell cycle have been to be inhibited by p21 K. J. I.B. Sci. S. A. 2000; PubMed Scopus Google Scholar, J. I.B. 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A. 2000; PubMed Scopus (83) Google Scholar). has been reported that mice hematopoietic and of cells M. J. A. S. P. M. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The of in K562 cells thus them toward erythroid differentiation in to inhibition by However, both p21 and p27 inhibit the that the distinct differentiation lineages by p21 and p27 on activities unrelated to cell cycle expression the of which is (12Denicourt C. Dowdy S.F. Genes Dev. 2004; 18: 851-855Crossref PubMed Scopus (157) Google Scholar, 13Coqueret O. Trends Cell Biol. 2003; 13: 65-70Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 2000; Google Scholar). p21 and p27 are highly similar in their Cdk inhibitory the for the of is not be that other proteins with complexes not with complexes and the inhibition by not the other of such a is the which the inhibitory of p21 on cyclin activity J.M. M. O. A. N. O. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the lines is possible that the as the proteins and with the cell is that the effects of p21 and p27 on Cdk activity are cell high differences in the interactions between CKIs and complexes example, p21 only the cyclin in the whereas p27 with both the cyclin and the Cdk thus significantly kinase activity. In the of the mechanisms by which p21 and p27 exert distinct effects on complexes and is for the in the differentiation further are to and for and for and for and for