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Cyclin D3 Regulates Proliferation and Apoptosis of Leukemic T Cell Lines

1999; Elsevier BV; Volume: 274; Issue: 49 Linguagem: Inglês

10.1074/jbc.274.49.34676

1083-351X

G. J. J. C. Boonen, Brigitte A. van Oirschot, Angela van Diepen, Wendy J.M. Mackus, Leo F. Verdonck, Gert Rijksen, René H. Medema,

Chronic Myeloid Leukemia Treatments

Activation of the T cell receptor in leukemic T cell lines or T cell hybridomas causes growth inhibition. A similar growth inhibition is seen when protein kinase C is activated through addition of phorbol myristate acetate. This inhibition is due to an arrest of cell cycle progression in G1 combined with an induction of apoptosis. Here we have investigated the mechanism by which these stimuli induce inhibition of proliferation in Jurkat and H9 leukemic T cell lines. We show that expression of cyclin D3 is reduced by each of these stimuli, resulting in a concomitant reduction in cyclin D-associated kinase activity. This reduction in cyclin D3-expression is crucial to the observed G1 arrest, since ectopic expression of cyclin D3 can abrogate the G1 arrest seen with each of these stimuli. Moreover, ectopic expression of cyclin D3 also prevents the induction of programmed cell death by phorbol myristate acetate and T-cell receptor activation, leading us to conclude that cyclin D3 not only plays a crucial role in progression through the G1 phase, but is also involved in regulating apoptosis of T cells. Activation of the T cell receptor in leukemic T cell lines or T cell hybridomas causes growth inhibition. A similar growth inhibition is seen when protein kinase C is activated through addition of phorbol myristate acetate. This inhibition is due to an arrest of cell cycle progression in G1 combined with an induction of apoptosis. Here we have investigated the mechanism by which these stimuli induce inhibition of proliferation in Jurkat and H9 leukemic T cell lines. We show that expression of cyclin D3 is reduced by each of these stimuli, resulting in a concomitant reduction in cyclin D-associated kinase activity. This reduction in cyclin D3-expression is crucial to the observed G1 arrest, since ectopic expression of cyclin D3 can abrogate the G1 arrest seen with each of these stimuli. Moreover, ectopic expression of cyclin D3 also prevents the induction of programmed cell death by phorbol myristate acetate and T-cell receptor activation, leading us to conclude that cyclin D3 not only plays a crucial role in progression through the G1 phase, but is also involved in regulating apoptosis of T cells. T cell receptor interleukin cyclin-dependent kinase phorbol myristate acetate antigen-induced cell death retinoblastoma protein phosphate-buffered saline polyacrylamide gel electrophoresis green fluorescent protein monoclonal antibody Activation of resting T lymphocytes requires ligation of the T cell receptor (TCR)1 and a co-stimulatory signal, provided by IL-2 or co-ligation of CD28 (reviewed in Ref. 1Lenschow D.J. Walunas T.L. Bluestone J.A. Annu. Rev. Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2367) Google Scholar). The combination of these stimuli enables the resting lymphocytes to exit G0 and proliferate. Remarkably, stimulation of the same TCR in T cells that are actively proliferating results in cessation of proliferation and subsequent apoptosis (2Smith C.A. Williams G.I. Kingston R. Jenkinson E.J. Owen J.J.T. Nature. 1989; 337: 181-183Crossref PubMed Scopus (1112) Google Scholar, 3Jones L.A. Chin L.T. Longo D.L. Kruisbeek A.M. Science. 1990; 250: 1726-1729Crossref PubMed Scopus (202) Google Scholar, 4Lenardo M.J. Nature. 1991; 353: 858-861Crossref PubMed Scopus (960) Google Scholar, 5Rocha B. von Boehmer H. Science. 1991; 251: 1225-1228Crossref PubMed Scopus (608) Google Scholar). This particular response is evident in thymocytes, but has also been described in leukemic T cells and T cell hybridomas (2Smith C.A. Williams G.I. Kingston R. Jenkinson E.J. Owen J.J.T. Nature. 1989; 337: 181-183Crossref PubMed Scopus (1112) Google Scholar, 6Ashwell J.D. Cunningham R.E. Noguchi P.D. Hernandez D. J. Exp. Med. 1987; 165: 173-194Crossref PubMed Scopus (211) Google Scholar, 7Mercep M.J. Bluestone J.A. Noguchi P.D. Ashwell J.D. J. Immunol. 1988; 140: 324-330PubMed Google Scholar, 8Takahashi S. Maecker H.T. Levy R. Eur. J. Immunol. 1989; 19: 1911-1919Crossref PubMed Scopus (65) Google Scholar). This indicates that the signals from the activated TCR complex can affect cell proliferation very differently, depending on the proliferative state of the T cell. Cell cycle progression from G0 to S phase in normal T cells requires the induction of the D-type cyclins (9Ando K. Ajchenbaum-Cymbalista F. Griffin J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9571-9575Crossref PubMed Scopus (170) Google Scholar). These cyclins are not expressed in resting T cells, but are induced upon activation of the TCR complex (10Ajchenbaum F. Ando K. DeCaprio J.A. Griffin J.D. J. Biol. Chem. 1993; 268: 4113-4119Abstract Full Text PDF PubMed Google Scholar). Three D-type cyclins have been identified, of which cyclins D2 and D3 are predominantly expressed in T cells (11Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Crossref PubMed Scopus (741) Google Scholar, 12Tam S.W. Theodoras A.M. Shay J.W. Draetta G.F. Pagano M. Oncogene. 1994; 9: 2663-2674PubMed Google Scholar). Cyclin D2 and D3 can form an active kinase complex with the cyclin-dependent kinase (cdk) 4 or cdk6, and the resulting kinase complexes are involved in phosphorylation of the retinoblastoma protein (pRb) (11Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Crossref PubMed Scopus (741) Google Scholar, 13Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1027) Google Scholar). Phosphorylation of pRb results in its functional inactivation and allows progression of the cell through the late G1 restriction point and subsequent entry into S phase (reviewed in Ref. 14Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4326) Google Scholar). However, cyclin D-cdk complexes are able to phosphorylate pRb only partially and complete phosphorylation and functional inactivation of pRb requires additional phosphorylation by cyclin E-cdk2 complexes (15Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (859) Google Scholar). Although cyclin D- and cyclin E-cdk complexes seem to phosphorylate overlapping sites in pRb, it has been demonstrated that some sites, such as Ser780, are only phosphorylated by cyclin D-cdk complexes (16Kitagawa M. Higashi H. Jung H-K. Suzuki-Takahashi I. Ikeda M. Tamai K. Kato J-Y. Segawa K. Yoshida E. Nishimura S. Taya Y. EMBO J. 1996; 15: 7060-7069Crossref PubMed Scopus (534) Google Scholar). Induction of cyclin D expression alone is not sufficient to drive resting T cells into S phase. This is due to the fact that resting T cells express abundant amounts of the cdk inhibitor p27kip1, that associates to the formed cyclin D-cdk complexes and inhibits their kinase activity (17Firpo E.J. Koff A. Solomon M.J. Roberts J.M. Mol. Cell. Biol. 1994; 14: 4889-4901Crossref PubMed Scopus (275) Google Scholar). Expression of p27kip1 is down-regulated as T cells progress through G1, but this process requires the presence of a co-stimulatory signal supplied by IL-2 (17Firpo E.J. Koff A. Solomon M.J. Roberts J.M. Mol. Cell. Biol. 1994; 14: 4889-4901Crossref PubMed Scopus (275) Google Scholar, 18Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M-H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar). Thus, only the combination of TCR activation and costimulation with IL-2 allows for the formation of active cyclin D-cdk complexes and initiation of DNA replication. Down-regulation of p27kip1 in normal T cells can be prevented by immunosuppressive agents such as rapamycin and cyclosporin, but also by elevation of intracellular cAMP levels (18Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M-H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar, 19Kato J.M. Matsuoka M. Polyak K. Massague J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar). As such these agents can prevent the appearance of active cyclin D-cdk complexes and suppress the proliferation of immunoreactive T cells. As mentioned above, the effect of TCR activation in actively proliferating T cells is dramatically different from that observed in resting T cells. In proliferating T cells, including leukemic T cells, engagement of the TCR complex results in a cell cycle arrest in G1 and TCR antigen-induced cell death (AID) (2Smith C.A. Williams G.I. Kingston R. Jenkinson E.J. Owen J.J.T. Nature. 1989; 337: 181-183Crossref PubMed Scopus (1112) Google Scholar, 3Jones L.A. Chin L.T. Longo D.L. Kruisbeek A.M. Science. 1990; 250: 1726-1729Crossref PubMed Scopus (202) Google Scholar, 4Lenardo M.J. Nature. 1991; 353: 858-861Crossref PubMed Scopus (960) Google Scholar, 5Rocha B. von Boehmer H. Science. 1991; 251: 1225-1228Crossref PubMed Scopus (608) Google Scholar, 6Ashwell J.D. Cunningham R.E. Noguchi P.D. Hernandez D. J. Exp. Med. 1987; 165: 173-194Crossref PubMed Scopus (211) Google Scholar, 7Mercep M.J. Bluestone J.A. Noguchi P.D. Ashwell J.D. J. Immunol. 1988; 140: 324-330PubMed Google Scholar, 8Takahashi S. Maecker H.T. Levy R. Eur. J. Immunol. 1989; 19: 1911-1919Crossref PubMed Scopus (65) Google Scholar). Such T cell activation can be mimicked by directly activating one of the downstream signaling pathways of the TCR complex. For example, activation of protein kinase C by adding PMA, either alone or in combination with calcium ionophores can cause growth inhibition and apoptosis in Jurkat T cells (2Smith C.A. Williams G.I. Kingston R. Jenkinson E.J. Owen J.J.T. Nature. 1989; 337: 181-183Crossref PubMed Scopus (1112) Google Scholar, 20Wyllie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 67-77Crossref PubMed Scopus (1443) Google Scholar, 21Shi Y. Bisonette R.P. Padrey N. Szalay M. Kubo R.T. Green D.R. J. Immunol. 1991; 146: 3340-3346PubMed Google Scholar). AID was recently shown to occur from a late G1 checkpoint in a Rb-dependent fashion (22Lissy N.A. Van Dyk L.F. Becker-Hapak M. Vocero-Akbani A. Mendler J.H. Dowdy S.F. Immunity. 1998; 8: 57-65Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Consistent with this notion is the finding that cyclin D3 is down-regulated by activation of the TCR in a T cell hybridoma (23Miyatake S. Nakano H. Park S.Y. Yamazaki T. Takase K. Matsushime H. Kato A. Saito T. J. Exp. Med. 1995; 182: 401-408Crossref PubMed Scopus (33) Google Scholar). However, the mechanism by which leukemic T cell lines arrest in G1 has not been elucidated. Therefore, we set out to investigate the effect of TCR activation on the expression and activity of a variety of cell cycle regulatory proteins known to be involved in progression through G1. In this report we show that TCR activation in Jurkat or H9 leukemic T cells results in a G1 arrest, associated with hypophosphorylated pRb. We show that expression of cyclin D3 is down-regulated in these cells, resulting in loss of phosphorylation of the Ser780residue on pRb. Very similar effects are seen upon stimulation with PMA. More importantly, ectopic expression of cyclin D3 cannot only overcome the growth inhibition induced by these stimuli, but also prevents the induction of apoptosis in response to these stimuli. This indicates that down-regulation of cyclin D3 is crucial to both the proliferative arrest as well as the induction of apoptosis seen with these stimulatory agents. Jurkat (E6.1, ATCC) and H9 (a kind gift of Dr. J.P. Medema, Leiden University) leukemic T cells were routinely cultured in RPMI 1640 medium (Life Technologies, Inc., Paisley, Scotland) supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Logan, Utah), l-glutamine (4 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), and 5 mm β-mercaptoethanol. Anti-CD3 mAb OKT3 and anti-CD4 mAb OKT4 (isotype control) were purified from hybridoma culture supernatant using protein A-Sepharose columns, anti-CD3 mAb UCHT-1 and anti-pRb (G3–245) were obtained from Pharmingen (Hamburg, Germany), anti-p27kip1 (clone 57) from Transduction Laboratories (Lexington, KY), anti-cyclin D3 (Ab-2) from Calbiochem (San Diego, CA), anti-cdk2 (M2) and anti-cyclin E (HE-12 for immunoblotting, HE-111 for immunoprecipitation and in vitrokinase activity) were all from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Ser780 was a kind gift of Dr. M. Kitagawa (16Kitagawa M. Higashi H. Jung H-K. Suzuki-Takahashi I. Ikeda M. Tamai K. Kato J-Y. Segawa K. Yoshida E. Nishimura S. Taya Y. EMBO J. 1996; 15: 7060-7069Crossref PubMed Scopus (534) Google Scholar) and anti-cyclin D2 (DCS-5) (24Lukas J. Bartkova J. Welcker M. Petersen O.W. Peters G. Strauss M. Bartek J. Oncogene. 1995; 10: 2125-2134PubMed Google Scholar) was a kind gift from Dr. J. Lukas (Danish Cancer Society, Copenhagen, Denmark). Propidium iodide was purchased from Sigma and protein A/G-Sepharose beads from Santa Cruz Biotechnology. For treatment with anti-CD3 mAbs or anti-CD4 mAb, tissue culture plates (96- or 12-well) were coated overnight at room temperature with 10 μg/ml anti-CD3 (OKT3 or UCHT-1) or 10 μg/ml anti-CD4 (OKT4), respectively, in phosphate-buffered saline (PBS). Control plates were treated overnight with PBS without any added mAbs. The coating solution was aspirated the next day and plates were washed three times with PBS. Plates were then immediately used for cell stimulation. For stimulation with PMA, cells were plated in the presence of 50 nm PMA. All cells were cultured in flasks in fresh medium at a density of 2 × 105cells/ml at 24 h prior to cell stimulation, to ensure that they were exponentially growing at the time of stimulation. Cells were then counted the next day and replated in the presence or absence of the different stimuli at 2 × 105 cells/ml. For analysis of cell cycle distribution, anti-CD3- or PMA-stimulated cells were collected at the indicated time points and washed with ice-cold PBS, after which they were fixed overnight in 70% ethanol at 4 °C. Cells were then pelleted by centrifugation and cell pellets were washed once with ice-cold PBS. Cells were stained with propidium iodide as described (25Medema R.H. Klompmaker R. Smits V.A.J. Rijksen G. Oncogene. 1998; 16: 431-441Crossref PubMed Scopus (151) Google Scholar). Cell cycle profiles were determined using a FACScalibur (Becton Dickinson) and analyzed using Cell Quest software. To determine [3H]thymidine incorporation, cells were plated at a density of 2 × 105 cells/ml in 96-well plates and cultured for the indicated times in the presence of the indicated stimuli. 1 μCi of [3H]thymidine was then added to each well, and cells were cultured for another 2 h. Cells were harvested and [3H]thymidine incorporation was determined in a scintillation counter. Cell viability after stimulation was determined by staining whole cells with propidium iodide, followed by flow cytometric analysis on a FACScalibur using Cell Quest software. Jurkat T cells were transfected by electroporation with a Gene Pulser (Bio-Rad) set at 260 V, 960 microfarads. For analysis of cell cycle profiles cells were transfected with 2 μg of pCMV.EGFP-spectrin (a kind gift of Dr. T. Shenk and Dr. A. J. Beavis) (26Kaletja R.F. Shenk T. Beavis A.J. Cytometry. 1997; 29: 286-291Crossref PubMed Scopus (111) Google Scholar) in combination with 20 μg of an expression vector for human cyclin D3 (pRcCMV-cyclin D3, a kind gift of Dr. P. Hinds, Harvard Medical School, Boston, MA). One day after transfection, viable cells were isolated by standard gradient centrifugation on Ficoll-Paque (Amersham Pharmacia Biotech). The cell suspension was then brought to a concentration of 2 × 105 viable cells/ml and used for stimulation in 12-well tissue culture plates. 24 h after stimulation cells were prepared for, and analyzed by flow cytometry as described (27Medema R.H. Herrera R.E. Lam F. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 91: 6289-6293Crossref Scopus (411) Google Scholar). To obtain Jurkat T cells stably expressing cyclin D3 the pRcCMV-cyclin D3 construct was introduced in Jurkat cells by electroporation and 2 days later the medium was changed to RPMI supplemented with 10% fetal calf serum and 2 mg/ml G418. Clonal lines were obtained by limiting dilution resulting in the isolation of JD3-I and JD3-II. After stimulation, cells were washed once with ice-cold PBS and cell pellets were resuspended and lysed in ELB lysis buffer (25Medema R.H. Klompmaker R. Smits V.A.J. Rijksen G. Oncogene. 1998; 16: 431-441Crossref PubMed Scopus (151) Google Scholar). Protein concentrations were determined and equal amounts of protein were used for immunoprecipitation with anti-cyclin E or anti-cdk2 antibodies. The immunoprecipitates were then used for in vitro kinase assays as described previously (25Medema R.H. Klompmaker R. Smits V.A.J. Rijksen G. Oncogene. 1998; 16: 431-441Crossref PubMed Scopus (151) Google Scholar). After proper stimulation, cells were harvested and washed once with ice-cold PBS. Cell pellets were then lysed in ELB buffer. Lysates were centrifuged, supernatants were collected, and protein concentrations were determined using the Bio-Rad protein assay. Samples were then separated on appropriate SDS-PAGE gels and blotted to nitrocellulose membrane. Proteins were then detected by ECL using standard protocols and appropriate antibodies. Activation of the TCR causes inhibition of proliferation of a variety of leukemic T cell lines, as well as different T cell hybridomas (2Smith C.A. Williams G.I. Kingston R. Jenkinson E.J. Owen J.J.T. Nature. 1989; 337: 181-183Crossref PubMed Scopus (1112) Google Scholar, 3Jones L.A. Chin L.T. Longo D.L. Kruisbeek A.M. Science. 1990; 250: 1726-1729Crossref PubMed Scopus (202) Google Scholar, 4Lenardo M.J. Nature. 1991; 353: 858-861Crossref PubMed Scopus (960) Google Scholar, 5Rocha B. von Boehmer H. Science. 1991; 251: 1225-1228Crossref PubMed Scopus (608) Google Scholar, 6Ashwell J.D. Cunningham R.E. Noguchi P.D. Hernandez D. J. Exp. Med. 1987; 165: 173-194Crossref PubMed Scopus (211) Google Scholar, 7Mercep M.J. Bluestone J.A. Noguchi P.D. Ashwell J.D. J. Immunol. 1988; 140: 324-330PubMed Google Scholar, 8Takahashi S. Maecker H.T. Levy R. Eur. J. Immunol. 1989; 19: 1911-1919Crossref PubMed Scopus (65) Google Scholar). We set out to study the mechanism by which this growth inhibition occurs in leukemic T cell lines. To this end, Jurkat and H9 leukemic T cell lines were stimulated by engaging the TCR complex with immobilized anti-CD3 mAbs. Treatment of both cell lines with the anti-CD3 mAb UCHT-1 for 24 h resulted in marked inhibition of proliferation, as measured by [3H]thymidine incorporation (Fig. 1 A). Growth inhibition was also observed with immobilized OKT3, another anti-CD3 mAb or phytohemagglutinin both known to efficiently stimulate signaling by the TCR complex (Fig. 1 A). Stimulation with an immobilized, isotype-matched anti-CD4 mAb or immobilized anti-CD28 mAb had no effect on cell proliferation (Fig. 1 A). Activation of protein kinase C, by the addition PMA, also resulted in efficient inhibition of cell proliferation (Fig. 1 A). Engagement of the TCR complex in leukemic T cells can result in apoptosis. Induction of apoptosis is also observed when the downstream signaling molecules protein kinase C and Ca2+-dependent calcineurin are directly activated through addition of phorbol esters and calcium ionophores. To determine the contribution of increased cell death to the growth inhibition observed in the Jurkat and H9 cells, we determined cell viability after stimulation with anti-CD3 mAb or PMA. We found an increase in the percentage of dead cells after stimulation with anti-CD3 mAb UCHT-1, from 3% in the unstimulated population to 13% after 24 h of stimulation and 22% after 48 h (Fig.1 B). When PMA was used as the stimulatory agent, we observed a similar increase in cell death, up to 29% after 24 h and a similar percentage after 48 h (Fig. 1 B). This would suggest that Jurkat and H9 cells do execute a program of AID in response T cell activation, which could explain the observed growth inhibition. However, the extent of cell death is not sufficient to fully explain the observed growth inhibition, since [3H]thymidine incorporation was inhibited by >50% after 24 h of stimulation with UCHT-1, whereas the amount of cell death was less than 15%. Addition of an anti-Fas mAb (Fas18) to these cells, triggered a rapid and dramatic cell death (>70% after 24 h) in these cell lines (data not shown), indicating that the relatively low levels of cell death observed after stimulation with anti-CD3 or PMA are not the result of a resistance of these cells to Fas-induced cell death. We decided to study the proliferative arrest in more detail by analysis of the DNA profiles using propidium iodide staining and flow cytometry. As shown in Fig. 2, in the untreated Jurkat cells ∼41% of the cells are in the G1 phase, ∼31% S phase, and ∼27% G2/M phase. However, as early as 16 h after plating on immobilized anti-CD3 mAbs, the percentage of cells in the G1 phase rises to ∼50%. After 24 h the percentage of cells in the G1 phase is more than 60% and a corresponding decrease in the percentage of cells in S phase is observed. Similar effects were seen in the H9 cell line (data not shown). Apoptotic cells undergo DNA fragmentation and therefore display a sub-G1 (<2N) DNA content. As can been seen in Fig. 2, the fraction of cells with a <2N DNA content is around 14% after 24 h of stimulation (Fig. 2). This indicates that the increased cell death observed after stimulation with anti-CD3 mAbs is at least partly due to induction of apoptosis, consistent with previous reports (8Takahashi S. Maecker H.T. Levy R. Eur. J. Immunol. 1989; 19: 1911-1919Crossref PubMed Scopus (65) Google Scholar, 28Zhu L. Anasetti C. J. Immunol. 1995; 154: 192-200PubMed Google Scholar). After stimulation with PMA, a similar increase in the percentage of cells in G1 is observed, up to 58% G1 cells after 48 h. Also, there was a clear increase in the fraction of apoptotic cells (<2N), from 1% in the unstimulated cells to 20% in the cells stimulated for 24 h with PMA. As mentioned above, a critical event during passage through G1 is the phosphorylation of pRb. To investigate whether the G1arrest occurred early, or late in G1, we examined the phosphorylation state of pRb after treatment of cells with immobilized anti-CD3 mAb or PMA for different time periods. In untreated asynchronous cultures, pRb is mostly present in its slower migrating hyperphosphorylated form (Fig.3 A). After 24 h of stimulation with the anti-CD3 mAb UCHT-1 or PMA we observed a clear mobility shift in the pRb protein from the hyperphosphorylated to the hypophosphorylated form (Fig. 3 A). We also measured the kinase activity of cyclin E-cdk2 complexes that are normally induced late in G1 and are involved in phosphorylation of pRb. After 24 h with anti-CD3 mAbs or PMA, cyclin E-associated kinase activity was inhibited by 22 or 38%, respectively (Fig. 3 B), indicating that these cells still contain a considerable amount of cyclin E-associated kinase activity after stimulation. Thus, it seems unlikely that the moderate inhibition of cyclin E-associated kinase activity is responsible for the observed growth inhibition. Similar reductions in kinase activity were observed using immunoprecipitates prepared with anti-cdk2 in in vitrokinase reactions (Fig. 3 C). Activation of G1 cyclin-cdk complexes in normal T cells involves down-regulation of the cdk-inhibitor p27kip1(17Firpo E.J. Koff A. Solomon M.J. Roberts J.M. Mol. Cell. Biol. 1994; 14: 4889-4901Crossref PubMed Scopus (275) Google Scholar, 18Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M-H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar). Stimulation of expression of p27kip1 by TCR activation in leukemic T cells could result in inhibition of the kinase activity of cyclin E-cdk2 complexes. Therefore, we examined if TCR activation can induce expression of p27kip1 in Jurkat T cells. Whole cell lysates were prepared from control cells and from cells stimulated for 24 h with immobilized anti-CD3 mAb UCHT-1 or PMA. As shown in Fig. 4 A, expression of p27kip1 did not change significantly upon activation of the T cells with anti-CD3 mAb or PMA, indicating that another mechanism must be responsible for the observed inhibition of cyclin/cdk activity in these leukemic cells. We next decided to analyze the effect of TCR activation on expression of the G1 cyclins. In T cells the major D-type cyclins are D2 and D3, whereas D1 is not expressed (11Meyerson M. Harlow E. Mol. Cell. Biol. 1994; 14: 2077-2086Crossref PubMed Scopus (741) Google Scholar, 12Tam S.W. Theodoras A.M. Shay J.W. Draetta G.F. Pagano M. Oncogene. 1994; 9: 2663-2674PubMed Google Scholar), and therefore we examined expression of cyclins D2, D3, and cyclin E. Cells were stimulated with immobilized anti-CD3 mAb or PMA for 24 h, since pRb was efficiently dephosphorylated at this time point (Fig.3 A). Expression of cyclin D3 was dramatically reduced after 24 h in the presence of PMA or immobilized anti-CD3 mAb in Jurkat cells (Fig. 4 B), suggesting that a reduction in cyclin D3 expression could be responsible for the observed G1 arrest. Cyclin D2 could not be detected in lysates from Jurkat (Fig.4 C) or H9 cells (not shown), in contrast to a control lysate from U2OS osteosarcoma cells shown previously to express cyclin D2 (24Lukas J. Bartkova J. Welcker M. Petersen O.W. Peters G. Strauss M. Bartek J. Oncogene. 1995; 10: 2125-2134PubMed Google Scholar). This indicates that cyclin D3 is the major D-type cyclin expressed in these leukemic T cell lines. Expression of cyclin E or its kinase partner cdk2 was unaltered upon stimulation with anti-CD3 mAb or PMA (Fig. 4, D and E), demonstrating that regulation of cyclin E or cdk2 expression is not involved in the inhibition of cyclin E/cdk2 kinase activity. In order to determine the timing of cyclin D3 down-regulation, we performed a time course experiment and total lysates were prepared for analysis of cyclin D3 expression levels. The timing of cyclin D3 down-regulation appears to be similar after stimulation with PMA or immobilized anti-CD3 mAbs. The reduction in cyclin D3 expression is first visible after 8 h of stimulation and continues to decline up to 24 h (Fig. 5 A). We next wanted to show that the reduction in cyclin D3 expression results in loss of cyclin D-associated kinase activity from the cell. Recently, it was shown that the Ser780 residue in pRb is phosphorylated by cyclin D-cdk complexes, but not by cyclin E- or cyclin A-cdk2 complexes (16Kitagawa M. Higashi H. Jung H-K. Suzuki-Takahashi I. Ikeda M. Tamai K. Kato J-Y. Segawa K. Yoshida E. Nishimura S. Taya Y. EMBO J. 1996; 15: 7060-7069Crossref PubMed Scopus (534) Google Scholar). Phosphorylation of Ser780 is therefore a good measure of cyclin D-cdk kinase activity without interference of the activity of other G1 cyclin-cdk complexes. Thus, we decided to study the phosphorylation state of the Ser780 residue of pRb after stimulation with PMA or anti-CD3 mAbs. For this purpose we made use of an antibody recognizing only pRb phosphorylated on Ser780(anti-phospho-Ser780) (16Kitagawa M. Higashi H. Jung H-K. Suzuki-Takahashi I. Ikeda M. Tamai K. Kato J-Y. Segawa K. Yoshida E. Nishimura S. Taya Y. EMBO J. 1996; 15: 7060-7069Crossref PubMed Scopus (534) Google Scholar). As shown in Fig. 5 B, treatment of Jurkat cells with PMA or immobilized UCHT-1 for 24 h resulted in loss of phosphorylation of pRb-Ser780. This clearly demonstrates that the reduction in cyclin D3 expression results in loss of cyclin D-associated kinase activity from the cell. Moreover, Ser780 phosphorylation was reduced as early as 12 h after stimulation with UCHT-1, indicating that the reduction in cyclin D3-associated kinase activity closely follows the decrease in cyclin D3 expression (Fig. 5 C). To determine if down-regulation of cyclin D3 is crucial to the proliferative arrest seen in response to PMA and anti-CD3 mAbs, we analyzed the effect of ectopic expression of cyclin D3 in Jurkat T cells. For this purpose, Jurkat cells were transiently transfected with an expression vector for cyclin D3. To allow analysis of the transfected population we co-transfected a plasmid encoding a modified version of the green fluorescent protein (GFP) (26Kaletja R.F. Shenk T. Beavis A.J. Cytometry. 1997; 29: 286-291Crossref PubMed Scopus (111) Google Scholar). Using bivariate flow cytometry we could then analyze the cell cycle distribution of the GFP-positive population (26Kaletja R.F. Shenk T. Beavis A.J. Cytometry. 1997; 29: 286-291Crossref PubMed Scopus (111) Google Scholar). Transfection of an empty vector control did not affect the growth arrest induced by anti-CD3 mAbs or PMA, as can be seen from the increase in the percentage of cells in the G1 phase after treatment with these agents (Fig. 6 A). However, transfection of the cyclin D3 expression plasmid resulted in an efficient rescue of the cell cycle arrest that normally occurs in response to treatment with anti-CD3 mAbs or PMA (Fig. 6 A), indicating that down-regulation of cyclin D3 is instrumental to the observed growth inhibition. To further corroborate these data, we established cell lines of Jurkat T cells stably overexpressing cyclin D3. We isolated two independent clones, JD3.I and JD3.II, and these lines were tested for growth inhibition by anti-CD3 mAbs and PMA. As can be seen in Fig.6 B, growth inhibition of JD3.I cells by UCHT-1 was reduced approximately 2-fold, and the response to PMA was almost completely abolished. The effect of cyclin D3 overexpression was even more pronounced in the JD3.II clone which expressed slightly higher levels of cyclin D3 (not shown). JD3.II cells were hardly inhibited in their growth by TCR activation or PMA treatment. To confirm that cyclin D3 was indeed constitutively expressed in these cell lines, we analyzed cyclin D3 expression on Western blots. Whereas cyclin D3 is efficiently down-regulated in the parental Jurkat cell line, no effect on cyclin D3 expression by anti-CD3 mAbs or PMA is seen in JD3.I (data not shown) or JD3.II (Fig. 6 C). Thus cyclin D3 can no longer be down-regulated in these cell lines by TCR activation and as a consequence no growth inhibition is observed. We then wanted to know if this rescue from growth inhibition had any influence on the apoptotic program induced by these stimuli. Interestingly, cell viability was no longer decreased in these cell lines after treatment with anti-CD3 mAbs or PMA (Fig. 6 D). We confirmed this by analysis of DNA profiles of the JD3.I and JD3.II cell lines after stimulation with UCHT-1 or PMA. Only a very small increase (∼6%) in the G1 percentage was seen in these cells after stimulation with UCHT-1 (Fig. 7), compared with an increase of 21% seen in normal Jurkat (see Fig. 2). The effect of PMA appeared to be completely abolished in JD3.I as well as JD3.II (Fig.7). Moreover, very little (1%) apoptotic cells were detected, confirming the data on cell viability obtained with these same cell lines. These data indicate that in addition to the growth arrest, AID also requires the down-regulation of cyclin D3. In this report we have addressed the mechanism by which TCR activation induces a G1 arrest in leukemic T cells. T cell activation was performed by stimulation with immobilized anti-CD3 mAbs. Under our experimental conditions, treatment with anti-CD3 mAbs resulted in a G1 arrest, and a reduction in cell viability. In parallel, cells were stimulated with the phorbol ester PMA, providing a somewhat more efficient stimulus. Activation of the same leukemic T cell lines with PMA induced a similar arrest in G1 and a similar reduction in cell viability. The G1 arrest induced by anti-CD3 mAbs and PMA was associated with the appearance of dephosphorylated pRb, indicating an arrest at, or prior to, the G1 restriction point. In agreement with this notion was the finding that the kinase activity of cyclin E and cdk2 complexes was reduced after TCR activation. In order to address the mechanism by which the reduction in kinase activity of these complexes occurs, we analyzed expression of the various G1 cyclins. Expression of cyclin E is usually induced somewhere late in G1 as a function of cell cycle progression. No reduction in the expression level of cyclin E could be detected after treatment with anti-CD3 mAbs, indicating that the inhibition of cyclin E-associated kinase occurs through active repression of the cyclin-cdk complex. Further analysis of expression of G1 cyclins showed that cyclin D2 is not expressed in Jurkat or H9 cells and that the expression of cyclin D3 was reduced significantly after treatment with anti-CD3 mAbs or PMA. At present we do not know whether the reduction in cyclin D3 expression occurs through active transcriptional repression or by a post-translational mechanism affecting protein stability of cyclin D3. The decrease in cyclin D3 expression resulted in a drop in the kinase activity of cyclin D-cdk complexes, as evidenced by the reduction in Ser780 phosphorylation of pRb seen after stimulation with PMA or anti-CD3 mAbs. Since p27kip1 was associated to some of those complexes the reduction in cyclin D3 levels will cause a rise in the amount of p27kip1 available to associate with cyclin E-cdk complexes. Therefore, the decrease in cyclin D3 expression might be indirectly responsible for the inhibition of cyclin E-cdk2 complexes. To investigate the importance of cyclin D3 down-regulation to the observed growth inhibition, we studied the effects of ectopic expression of cyclin D3. We show that overexpression of cyclin D3, both transiently as well as in stable cell lines, is sufficient to override the cell cycle arrest induced by TCR activation, indicating that the growth inhibition requires down-regulation of cyclin D3. In addition, ectopic expression of cyclin D3 prevents the apoptosis induced by stimulation with anti-CD3 mAbs or PMA. These data implicate that the down-regulation of cyclin D3 is not only crucial to the growth arrest, but also for apoptosis induced by TCR activation. This would suggest that cyclin D3 integrates signals that regulate the balance between proliferation and apoptosis of T cells. Since D-type cyclins are required for inactivation of pRb and it is known that cells lacking functional pRb no longer require cyclin D-associated kinase activity (23Miyatake S. Nakano H. Park S.Y. Yamazaki T. Takase K. Matsushime H. Kato A. Saito T. J. Exp. Med. 1995; 182: 401-408Crossref PubMed Scopus (33) Google Scholar, 29Lukas J. Parry D. Aagaard L. Mann D.J. Bartkova J. Strauss M. Peters G. Bartek J. Nature. 1995; 375: 503-506Crossref PubMed Scopus (875) Google Scholar, 30Koh J. Enders G.H. Dynlacht B.D. Harlow E. Nature. 1995; 375: 506-510Crossref PubMed Scopus (523) Google Scholar), one would expect that T cells that lack functional pRb are not inhibited in their growth by TCR activation. Indeed, Lissyet al. (22Lissy N.A. Van Dyk L.F. Becker-Hapak M. Vocero-Akbani A. Mendler J.H. Dowdy S.F. Immunity. 1998; 8: 57-65Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) recently reported that Jurkat T cells can be rescued from AID when pRb is functionally inactivated through introduction of the HPV E7 protein. They showed that AID occurs from a late G1 phase cell cycle checkpoint. Our data provide further insight into the underlying mechanism and suggest that this checkpoint is activated through down-regulation of cyclin D3. Expression of the D-type cyclins is low in cells in G0, such as resting T cells (10Ajchenbaum F. Ando K. DeCaprio J.A. Griffin J.D. J. Biol. Chem. 1993; 268: 4113-4119Abstract Full Text PDF PubMed Google Scholar). Upon stimulation of G0 cells with the proper mitogens, expression of cyclin D is induced, and this remains high as long as the mitogens are present (9Ando K. Ajchenbaum-Cymbalista F. Griffin J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9571-9575Crossref PubMed Scopus (170) Google Scholar). In normal peripheral T cells, induction of cyclin D3 expression requires TCR activation and this is further stimulated after addition of IL-2 (31Boonen G.J.J.C. van Dijk A.M.C. Verdonck L.F. van Lier R.A.W. Rijksen G. Medema R.H. Eur. J. Immunol. 1999; 29: 789-798Crossref PubMed Google Scholar). However, leukemic cells, such as the Jurkat cells used in this study, no longer depend on TCR activation or IL-2 for their proliferation and express constitutively high levels of cyclin D3. Remarkably, our data show that TCR activation has the opposite effect on cyclin D3 expression from that observed in normal peripheral T cells. Future experiments are called for to elucidate the mechanism by which TCR activation can cause the reduction in cyclin D3 levels. It will be of significant interest to learn why this mechanism only exists in actively proliferating T cells and how the effect of TCR activation on cyclin D3 expression can be so radically different in resting T cells. This could help us understand how control of cellular proliferation in leukemic T cells is different form normal T cells and lead to more insight into potential treatments of such malignancies. We thank Dr. Jiri Lukas (Danish Cancer Society, Copenhagen, Denmark) for the cyclin D2 antibodies, Dr. Masatoshi Kitagawa (Medical Institute of Bioregulation, Kyushu University, Japan) for the phosphospecific antibody recognizing Ser780-phosphorylated pRb, and Dr. Eric Lam (Ludwig Institute, London, UK) for helpful discussions. Also, we thank the other members of the Jordan Laboratory for helpful discussions.