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The Cell Cycle Inhibitor p16INK4A Sensitizes Lymphoblastic Leukemia Cells to Apoptosis by Physiologic Glucocorticoid Levels

2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês

10.1074/jbc.m008188200

1083-351X

Michael J. Ausserlechner, Petra Obexer, G. Jan Wiegers, Bernd Hartmann, Stephan Geley, Reinhard Kofler,

Retinoids in leukemia and cellular processes

The cyclin-dependent kinase inhibitor p16INK4A is frequently inactivated in childhood T-cell acute lymphoblastic leukemia. To investigate possible consequences of this genetic alteration for tumor development, we conditionally expressed p16INK4A in the T-cell acute lymphoblastic leukemia line CCRF-CEM, which carries a homozygous deletion of this gene. In agreement with its reported function, p16INK4A expression was associated with hypophosphorylation of the retinoblastoma protein pRB and stable cell cycle arrest in G0/G1, documenting that the pRB/E2F pathway is functional in these cells. Unexpectedly, p16INK4Aexpression increased the sensitivity threshold for glucocorticoid (GC)-induced apoptosis from therapeutic to physiologic levels. As a possible explanation for this phenomenon, we found that p16INK4A-arrested cells had elevated GC receptor expression associated with enhanced GC-mediated transcriptional activity and increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GC receptor levels critically influence GC sensitivity and imply that p16INK4Ainactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell death triggered by endogenous GC. The cyclin-dependent kinase inhibitor p16INK4A is frequently inactivated in childhood T-cell acute lymphoblastic leukemia. To investigate possible consequences of this genetic alteration for tumor development, we conditionally expressed p16INK4A in the T-cell acute lymphoblastic leukemia line CCRF-CEM, which carries a homozygous deletion of this gene. In agreement with its reported function, p16INK4A expression was associated with hypophosphorylation of the retinoblastoma protein pRB and stable cell cycle arrest in G0/G1, documenting that the pRB/E2F pathway is functional in these cells. Unexpectedly, p16INK4Aexpression increased the sensitivity threshold for glucocorticoid (GC)-induced apoptosis from therapeutic to physiologic levels. As a possible explanation for this phenomenon, we found that p16INK4A-arrested cells had elevated GC receptor expression associated with enhanced GC-mediated transcriptional activity and increased responsiveness of the GC-regulated cyclin D3 gene. These data are supported by our previous findings that GC receptor levels critically influence GC sensitivity and imply that p16INK4Ainactivation, in addition to allowing unrestricted proliferation, represents a mechanism by which lymphoid tumor cells might escape cell death triggered by endogenous GC. T-cell acute lymphoblastic leukemias cyclin-dependent kinase glucocorticoid glucocorticoid receptor phosphate-buffered saline murine mammary tumor virus 4-morpholinopropanesulfonic acid fluorescein isothiocyanate fluorescence-activated cell sorter The INK4A gene locus is located at chromosome 9p21 and encodes two different cell cycle inhibitors, namely p16INK4A and p19ARF (p14ARF), transcribed from alternative exons of the same gene, but dissimilar in their protein structures (1Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3420) Google Scholar, 2Quelle D.E. Zindy F. Ashmun R.A. Sherr C.J. Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1329) Google Scholar). Inactivation of this gene locus is observed in up to 80‥ of primary T-cell acute lymphoblastic leukemias (T-ALLs),1 thereby being the most consistent genetic alteration in this disease (3Cayuela J.M. Madani A. Sanhes L. Stern M.H. Sigaux F. Blood. 1996; 87: 2180-2186Crossref PubMed Google Scholar, 4Takeuchi S. Bartram C.R. Seriu T. Miller C.W. Tobler A. Janssen J.W. Reiter A. Ludwig W.D. Zimmermann M. Schwaller J. Blood. 1995; 86: 755-760Crossref PubMed Google Scholar, 5Hebert J. Cayuela J.M. Berkeley J. Sigaux F. Blood. 1994; 84: 4038-4044Crossref PubMed Google Scholar, 6Ohnishi H. Kawamura M. Ida K. Sheng X.M. Hanada R. Nobori T. Yamamori S. Hayashi Y. Blood. 1995; 86: 1269-1275Crossref PubMed Google Scholar). The high frequency in T-ALLs suggests that the tumor suppressor genes encoded by this locus might play an important role in the development of this malignancy. Ink4a knockout mice develop spontaneous lymphomas with high frequency, further suggesting that p16INK4A might be involved in negative regulation of proliferation in the lymphoid lineage (7Serrano M. Lee H. Chin L. Cordon C.C. Beach D. DePinho R.A. Cell. 1996; 85: 27-37Abstract Full Text Full Text PDF PubMed Scopus (1420) Google Scholar). p16INK4A acts by binding to and inhibiting the activity of CDK4 and CDK6 kinases (1Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3420) Google Scholar, 8Sherr C.J. Science. 1996; 274: 1672-1676Crossref PubMed Scopus (5016) Google Scholar). When complexed to regulatory cyclins of the D-type, these kinases phosphorylate and inactivate the retinoblastoma protein pRB and its family members, thereby releasing transcription factors, such as E2F-1, from inhibition by pRB. This allows transcription of genes essential for the onset of S phase (9Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3225) Google Scholar). To further investigate a possible role of p16INK4Ainactivation in T-ALL tumor development, we used the childhood T-ALL model CCRF-CEM, which contains a homozygous deletion of theINK4A locus (10Ogawa S. Hirano N. Sato N. Takahashi T. Hangaishi A. Tanaka K. Kurokawa M. Tanaka T. Mitani K. Yazaki Y. Hirai H. Blood. 1994; 84: 2431-2435Crossref PubMed Google Scholar, 11Otsuki T. Clark H.M. Wellmann A. Jaffe E.S. Raffeld M. Cancer Res. 1995; 55: 1436-1440PubMed Google Scholar), and the tetracycline-regulated gene expression system (12Loffler M. Ausserlechner M.J. Tonko M. Hartmann B.L. Bernhard D. Geley S. Helmberg A. Kofler R. Oncogene. 1999; 18: 4626-4631Crossref PubMed Scopus (26) Google Scholar) to conditionally express transgenic p16INK4A. p16INK4A expression was associated with a stable arrest in the G0/G1 phase of the cell cycle. Surprisingly, p16INK4A arrest dramatically increased the sensitivity of these cells to glucocorticoid (GC), a hormone that is routinely used for its apoptosis-inducing property in the therapy of this malignancy. Since p16INK4A increased the sensitivity of these leukemia cells to physiologic levels of the hormone, we speculate that p16INK4A inactivation (or a functionally equivalent mechanism) might be required for lymphoid malignancies to escape an as yet unrecognized tumor surveillance for lymphoid lineage,i.e. GC-induced apoptosis. CEM-C7H2 (13Strasser-Wozak E.M. Hattmannstorfer R. Hala M. Hartmann B.L. Fiegl M. Geley S. Kofler R. Cancer Res. 1995; 55: 348-353PubMed Google Scholar) is a highly GC-sensitive subclone of the CCRF-CEM-C7 cell line (14Norman M. Thompson E.B. Cancer Res. 1977; 37: 3785-3791PubMed Google Scholar). The generation and analysis of the CEM-C7H2 subclone C7H2-2C8, which contains the pβrtTA plasmid coding for the reverse tetracycline-responsive transactivator (15Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2069) Google Scholar), have been published (12Loffler M. Ausserlechner M.J. Tonko M. Hartmann B.L. Bernhard D. Geley S. Helmberg A. Kofler R. Oncogene. 1999; 18: 4626-4631Crossref PubMed Scopus (26) Google Scholar). All cells were maintained in RPMI 1640 medium containing 10‥ fetal calf serum (Life Technologies, Paisley, United Kingdom), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine (Life Technologies) at 5‥ CO2 and 37 °C in saturated humidity. Dexamethasone and cortisol were stored as 10 mm stock solutions in 100‥ ethanol; doxycycline was kept as a 10 mm solution dissolved in phosphate-buffered saline (PBS). All reagents were from Sigma (Vienna, Austria), unless indicated otherwise. For each experiment, mid-log phase cultures (2.5–5 × 105 cells/ml) were centrifuged and resuspended in fresh medium at a concentration of ∼2.5 × 105 cells/ml. Full-length p16INK4AcDNA was subcloned from pSK-p16INK4A (1Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3420) Google Scholar) into theEcoRI-XbaI sites of the pUHD10-3 vector for tetracycline-regulated expression (16Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4301) Google Scholar), generating plasmid pUHD10-3-p16INK4A. pKS-tkHyg is a pBluescript II-KS vector (Stratagene, La Jolla, CA) containing the hygromycin B resistance gene under the control of a thymidine kinase promoter and a SV40 polyadenylation site (17Hartmann B.L. Geley S. Loffler M. Hattmannstorfer R. Strasser-Wozak E.M.C. Auer B. Kofler R. Oncogene. 1999; 18: 713-718Crossref PubMed Scopus (47) Google Scholar). Transactivation reporter assays were performed using an MMTV-luciferase construct (18Drouin J. Sun Y.L. Chamberland M. Gauthier Y. De Lean A. Nemer M. Schmidt T.J. EMBO J. 1993; 12: 145-156Crossref PubMed Scopus (277) Google Scholar) cotransfected with the pRL-SV40 plasmid (Promega, Madison, WI) as a transfection control. For stable introduction of p16INK4A, C7H2-2C8 cells were transfected by electroporation as previously described (19Geley S. Hartmann B.L. Kapelari K. Egle A. Villunger A. Heidacher D. Greil R. Auer B. Kofler R. FEBS Lett. 1997; 402: 36-40Crossref PubMed Scopus (39) Google Scholar). Briefly, ∼1 × 107 mid-log phase cells in 800 μl of PBS containing 30 μg of linearized pUHD10-3-p16INK4A plasmid and 25 μg of pKS-tkHyg plasmid were electroporated at 300 V and 500 microfarads using a Bio-Rad Gene Pulsar. The cells were cultured in two 96-well plates for 2 days and then exposed to 0.25 mg/ml hygromycin B. Resistant transformants were selected and analyzed for conditional p16INK4A expression by Northern and Western blot analyses. Total RNA was extracted from 5 × 106 cells with TriReagentTM (LPS Industries, Moonachie, NJ). Approximately 10 μg of RNA were separated by electrophoresis on a denaturing 1‥ agarose gel containing formaldehyde in MOPS buffer and blotted overnight onto ZetabindTM nylon membranes (Cuno, Inc., Meriden, CO) according to standard protocols. RNA was cross-linked to the membranes by UV light. Filters were then prehybridized in phosphate blocking buffer containing SDS and bovine serum albumin at 65 °C for 3 h and hybridized for 12 h to a [32P]dATP-labeled, heat-denatured, full-length p16INK4A probe. After hybridization, blots were washed in 1× saline/sodium phosphate/EDTA and 0.1‥ SDS at 65 °C and in 0.1× saline/sodium phosphate/EDTA and 0.1‥ SDS at 65 °C and exposed to Agfa Curix x-ray films with an amplifying screen at −90 °C for several hours to days. After each hybridization, the blots were stripped by boiling in 0.1‥ SDS and rehybridized with a full-length glyceraldehyde-3-phosphate dehydrogenase probe. For quantification of apoptosis, nuclear staining with propidium iodide in concert with forward/sideward scatter analysis was used (20Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4447) Google Scholar). Briefly, cells were centrifuged, and the pellets were resuspended in 0.7 ml of hypotonic propidium iodide solution. The tubes were kept at 4 °C in the dark overnight. Nuclear fluorescence intensity and forward/sideward scatter were analyzed with a Becton Dickinson FACScan. Cell debris and small particles were excluded from analysis, and nuclei in the sub-G1 marker window were considered to represent apoptotic cells. Cells were washed in PBS and lysed for 30 min on ice in PBS lysis buffer containing 1‥ Nonidet P-40 and 10 mm sodium fluoride, to which 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin were added just before use. Cell lysates were cleared by centrifugation. An equal amount of 2× SDS sample buffer containing 10‥ β-mercaptoethanol was added, and proteins were denatured by boiling for 2 min. Samples were separated by SDS-polyacrylamide gel electrophoresis on 7.5–15‥ polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes by a Bio-Rad semidry transfer apparatus and stained using Ponceau red. The membranes were incubated with Tris-buffered saline blocking buffer containing 1‥ Tween 20 and 5‥ nonfat dry milk for 30 min, followed by an overnight incubation at 4 °C with primary antibody diluted in blocking buffer. Blots were probed with monoclonal or polyclonal antibodies against human cyclin D3, p16INK4A (Pharmingen, Hamburg, Germany), pRB, and α-tubulin (Oncogene Research, Cambridge, MA). Membranes were washed in Tris-buffered saline and incubated with a horseradish peroxidase-conjugated anti-mouse secondary antibody (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) diluted in blocking buffer. Finally, the blots were developed by the enhanced chemiluminescence substrate ECL (Amersham Pharmacia Biotech) according to the manufacturer's instructions and exposed to Agfa Curix x-ray films from seconds to several minutes. Stripping and reprobing were performed as described by the manufacturer. Cultured cells were washed twice in PBS and 1‥ bovine serum albumin. Aliquots of 1 × 106 cells were fixed in 1‥ paraformaldehyde at room temperature for 30 min, washed, and permeabilized with 0.1‥ Triton X-100 in 0.1‥ citrate buffer for 5 min on ice. Cells were washed twice and incubated at room temperature for 70 min in the presence of either FITC-conjugated anti-GR antibody 5E4 (a kind gift from Dr. A. Falus) (21Berki T. Kumanovics G. Kumanovics A. Falus A. Ujhelyi E. Nemeth P. J. Immunol. Methods. 1998; 214: 19-27Crossref PubMed Scopus (47) Google Scholar) or an FITC-conjugated isotype control (IgG1, Pharmingen). Finally, cells were washed three times, incubated with propidium iodide, resuspended in PBS and 1‥ bovine serum albumin, and analyzed on a FACScan. For determination of ligand binding by the GR, whole cell ligand binding assays were performed as described previously (22Helmberg A. Fassler R. Geley S. Johrer K. Kroemer G. Bock G. Kofler R. J. Immunol. 1990; 145: 4332-4337PubMed Google Scholar). Briefly, ∼5 × 106 cells were incubated in triplicate with increasing amounts of [3H]triamcinolone acetonide (PerkinElmer Life Sciences) in the presence or absence of a 500-fold molar excess of unlabeled triamcinolone acetonide at 37 °C for 1 h, washed three times, and resuspended in scintillation mixture (Packard Instrument Co., Groningen, The Netherlands). The samples were counted in a scintillation counter. For transient MMTV reporter transfections, 5 × 106 mid-log phase cells were washed in PBS and resuspended in 4 ml of RPMI 1640 medium containing 10‥ fetal calf serum. In each experiment, 5 μg of pMMTV-luc and 1 μg of pRL-SV40 DNA were transfected using SuperfectTM(QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. After a 3-h incubation, 15 ml of medium were added, and cells were split and incubated in the presence or absence of 200 ng/ml doxycycline for another 18 h. Cultures were then split again, and 100 nm dexamethasone was added for another 10 h to study specific induction of the MMTV reporter. Thereafter, the cells were harvested washed once in PBS, and cell pellets were lysed in 25 μl of reporter lysis buffer for 15 min at room temperature and centrifuged. The supernatant of each sample was analyzed using the dual-luciferase reporter system (Promega) according to the manufacturer's instructions. To determine whether the CDK4/6 inhibitor p16INK4A can induce cell cycle arrest in CCRF-CEM cells, we generated stably transfected CCRF-CEM derivatives with tetracycline-inducible p16INK4Aexpression. For this purpose, C7H2-2C8, a CEM-C7H2 subclone with constitutive reverse tetracycline-responsive transactivator expression (12Loffler M. Ausserlechner M.J. Tonko M. Hartmann B.L. Bernhard D. Geley S. Helmberg A. Kofler R. Oncogene. 1999; 18: 4626-4631Crossref PubMed Scopus (26) Google Scholar), was cotransfected with a plasmid expressing p16INK4Afrom a reverse tetracycline-transactivator-responsive promoter and a hygromycin resistance plasmid. Three hygromycin-resistant clones, referred to as 6E2/p16, 1D2/p16, and 1E10/p16, showed high levels of p16INK4A mRNA (Fig.1 A) and protein expression (Fig. 1 B) upon addition of the tetracycline analog doxycycline, but essentially no p16INK4A expression in its absence. The p16INK4A protein expression level was comparable to that found in aged human fibroblasts (data not shown). Two of these subclones, 6E2/p16 and 1D2/p16, were selected for further analysis. Induction of p16INK4A increased the electrophoretic mobility of pRB (Fig. 2), presumably by prevention of pRB phosphorylation. Cyclin D3, which forms a functional complex with CDK4 and CDK6, remained at unchanged levels. Twenty-four hours after addition of doxycycline, p16INK4A-expressing cells were completely arrested in the G0/G1 phase of the cell cycle, as demonstrated by FACS cell cycle analysis (Fig.3), suggesting that the downstream components of the p16INK4A/pRB pathway are intact. p16INK4A-mediated cell cycle arrest was maintained over 72 h without signs of reduced viability.Figure 2p16INK4A expression in stably transfected CCRF-CEM leukemia cells is associated with pRb hypophosphorylation. Parental C7H2-2C8 control (2C8/Ctr) and p16INK4A-transfected 6E2/p16 and 1D2/p16 cells were cultured in the presence or absence of 200 ng/ml doxycycline (Dox) for 24 h. Cytoplasmic extracts were analyzed by immunoblotting using monoclonal antibodies directed against p16INK4A, cyclin D3 (Cyc D3), pRB, and α-tubulin (α-Tub).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3p16INK4A expression in stably transfected CCRF-CEM leukemia cells is followed by a stable arrest in G0/G1. C7H2-2C8 control (2C8/Ctr) and p16INK4A-transfected 6E2/p16 and 1D2/p16 cells were cultured in the presence (+Dox) or absence (−Dox) of 200 ng/ml doxycycline for 36 h. The cells were subjected to cell cycle determination by FACS analysis of propidium iodide-stained nuclei.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since GCs are central components in the therapy of childhood T-ALL, we investigated whether p16INK4A expression might exert any effects on the sensitivity of these leukemia cells to GC-induced apoptosis. To this end, 6E2/p16, 1D2/p16, and parental C7H2-2C8 control cells were cultured for 24 h in the presence or absence of doxycycline and subsequently exposed to a 10 or 100 nm concentration of the therapeutic GC analog dexamethasone for another 24, 36, and 48 h. As already shown in Fig. 3, exposure to doxycycline induced cell cycle arrest in 6E2/p16 and 1D2/p16, but had no effect upon the parental C7H2-2C8 control line. When G0/G1-arrested cells, i.e. doxycycline-treated 6E2/p16 and 1D2/p16 cells, were treated with 100 nm dexamethasone, they exhibited accelerated apoptosis compared with cycling cells, starting within 24 h and reaching a plateau during the following 12 h. In contrast, doxycycline had no effect on the kinetics of cell death induced by 100 nm dexamethasone in parental C7H2-2C8 control cells (Fig. 4, left panels). Moreover, 10 nm dexamethasone (Fig. 4,right panels), which had no apoptotic effect on proliferating cells, was almost as efficient as 100 nmdexamethasone in cell death induction in G0/G1-arrested 6E2/p16 and 1D2/p16 cells. We next studied the sensitivity of these cells to the physiologic GC, cortisol. The parental 2C8 control line and its p16INK4A-transfected subclones (in the absence of doxycycline) were highly resistant to cortisol at concentrations up to 5000 nm, which is ∼100-fold higher than free cortisol in healthy humans (Fig.5 A). Surprisingly, massive apoptosis induction was observed in p16INK4A-expressing cells treated with as little as 50 nm cortisol (Fig.5 A). Apoptosis induction by physiologic cortisol in G0/G1-arrested cells was prevented by addition of RU486, suggesting that the observed effect was mediated by the GR (Fig. 5 B). Thus, p16INK4A expression sensitized the cells to physiologic concentrations of cortisol.