Leukemia (2001) 15, 1225–1231 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Comparison of the effects of recombinant adenovirus-mediated expression of wild- type p53 and p27Kip1 on cell cycle and apoptosis in SUDHL-1 cells derived from anaplastic large cell lymphoma F Turturro1,2, AY Frist1, MD Arnold1, A Pal1, GA Cook1 and P Seth3 1Human Gene Therapy Research Institute, John Stoddard Cancer Center, and 2Division of Hematology-Oncology, VA Central Iowa Health Care System, 3University Research, Des Moines University, Des Moines, IA, USA Recombinant adenoviruses expressing wild-type p53 family including proteins such as p21Waf1 and p57Kip2.11 CKD- Kip1 (AdWTp53) and p27 (Adp27) were used to compare the Is induce cell cycle arrest in G1 by inhibiting the kinase effects on cell cycle and apoptosis in SUDHL-1 cells derived activity ofcomplex cyclin E-CDK2. It has been shown that from human anaplastic large cell lymphoma. Cells infected with Kip1 AdWTp53 and Adp27 showed high level of wild-type p53 and the interaction ofp27 with CDK2-cyclin E complex blocks p27Kip1 expression, respectively. The expression of these pro- phosphorylation ofCDK2 on Thr 160 via a CDK activation teins resulted in G1 arrest after 24 h of infection. Although the kinase.11 Cyclic AMP and other negative regulators ofthe cell cells persisted in G1 arrest in both cell populations after 48 and cycle induce p27Kip1.12 The expression ofp27 Kip1 is downregu- 72 h of infection, the level of apoptosis assessed by TUNEL lated by mitogens.13 Inhibition ofthe p27 Kip1 expression with analysis was higher in cells infected with AdWTp53. Interest- ingly, apoptosis was more pronounced in cells infected with antisense oligonucleotides antagonizes the response to 14 Adp27 after the initial 24 h and reached a steady state at 48 mitogen depletion preventing cell cycle arrest. This indicates and 72 h. A lower MOI of Adp27 resulted in G1 arrest associated that the protein is an essential component ofthe pathway that with a low level of apoptosis in SUDHL-1 cells after 48 h of relates mitogenic signals to the cell cycle.14 Lloyd et al11 have infection. This was correlated with lower expression of p27Kip1. recently defined p27Kip1 a multifunctional protein and have We postulate that the time-lag and the different level of analyzed its putative functions of CDK inhibition, tumor sup- apoptosis occurring in SUDHL-1 cells infected with AdWTp53 and Adp27 are clearly related to the intrinsic biochemical path- pression, induction of apoptosis and cell differentiation. The Kip1 ways solicited. In this context our study provides a model to relevance ofp27 as a prognostic factor and as a tumor investigate these pathways and better understand the biology suppressor protein is underscored by the low incidence of of this particular lymphoma. Our data also support a potential gene mutations in human malignancies.15,16 application of Adp27 for gene therapy of this lymphoma simi- These studies prompted us to investigate whether the larly to AdWTp53 as previously shown. Leukemia (2001) 15, expression ofp27 Kip1 by an adenoviral vector (Adp27) exerted 1225–1231. Keywords: adenovirus; p53; p27Kip1; cyclin-dependent kinase a function similar to exogenously expressed wtp53 in inhibitor; cell cycle; apoptosis SUDHL1cells. Our intent was to evaluate the potential appli- cation ofAdp27 in a cellular model previously used for wtp53, a prototype oftumor suppressor. 2,7 Introduction Apoptosis is involved in growth and differentiation of multi- Materials and methods cellular systems. It has recently been shown that apoptosis follows a persistent G1 arrest during the cell cycle.1 The tumor Cell culture and recombinant adenovirus infection suppressor p53 plays an important role as trans-activator of 7 gene expression ofproteins involved in the regulation ofcell SUDHL-1 cells have been previously described. Cells were cycle arrest.2,3 Mutations that inactivate the p53 tumor sup- cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD, pressor gene are the most common genetic abnormalities in USA) containing 15% heat-inactivated FBS as previously 7 × 5 human malignancies. Genetically engineered p53-deficient described. SUDHL-1 cells (5 10 ) were infected as pre- mice develop spontaneous tumor.4 The tumor suppressor p53 viously described with 100 MOI ofAdNull (adenoviral vector backbone without c-DNA insert), AdWTp53 (expressing regulates the expression ofits target genes by binding to spe- Kip1 7,8,17 cific sequence ofDNA and by interacting with several wtp53) and Adp27 (expressing p27 ). Uninfected cells elements ofthe transcription complex. 1,5 One ofthe target were used as controls (mock). For some experiments 50 MOI proteins that mediates p53-induced cell cycle arrest is the ofAdNull and Adp27 were used. cyclin-dependent kinase inhibitor (CDK-I) p21Waf1.6 We have recently shown that wild-type p53 expressed by the adenovi- ral vector AdWTp53, induced apoptosis in SUDHL-1 cells, Western blot analysis derived from human anaplastic large cell lymphoma (ALCL).7 × 5 Several studies have recently demonstrated that adenoviral SUDHL-1 cells (5 10 ) were infected with recombinant vector-mediated expression ofp27 Kip1 induces cell cycle arrest adenoviruses as described above. Total proteins derived from and apoptosis in cell lines derived from epithelial malig- cells in each group ofinfectionwere separated on gradient nancies.8–10 The CDK-I p27Kip1 is a member ofthe Cip/Kip ready-made polyacrylamide gel (BioRad, Hercules, CA, USA) and transferred to nitrocellulose. Blots were incubated with blocking solution (5% dry milk in PBS/Tween 20) and then probed with primary antibodies anti-p53 (Ab-6, monoclonal; Correspondence: F Turturro, Human Gene Therapy Research Institute, 1415 Woodland Ave, Des Moines, IA, 50309–3203, USA; Fax +1 Oncogene Research Products, Cambridge, MA, USA) and 515–241–8788 anti-p27 (F-8, monoclonal; Santa Cruz Biotechnology, Santa Received 26 February 2001; accepted 23 April 2001 Cruz, CA, USA) for 1 h at room temperature. ␤-Actin antibody Cell cycle and apoptosis induced by exogenous expression of wild-type p53 and p27Kip1 F Turturro et al 1226 (AC-15; Sigma, St Louis, MO, USA) was used as control for some experiments. Blots were subsequently incubated with secondary anti-IgG-HRP conjugated antibody (Sigma). Blots were developed using ECL kit (Amersham Pharmacia, Piscata- way, NJ, USA) and exposed to X-ray film. Cell cycle analysis For DNA content analysis, cells were stained with propidium iodine (PI, Coulter Reagents Kit; Coulter, Miami, FL, USA), as previously described, and analyzed with an Epics cytometer (Coulter) in duplicate after 24, 48 and 72 h of infection.7 His- tograms ofthe percentage ofcells in the G1, G2 and S phase were expressed as mean ± s.d. Figure 1 Western blot analysis ofwtp53 and p27 Kip1 expression in SUDHL-1 cells after infection with AdWTp53 or Adp27. Cells were infected with 100 MOI of AdNull (adenovirus backbone) AdWTp53 or Adp27. Uninfected cells were used as controls (mock). After 24 h Flow cytometry analysis of apoptosis ofinfectioncell lysates were subjected to Western blot analysis with monoclonal antibody anti-p53 (a) and anti-p27 (b), as described in SUDHL-1 cells were stained for terminal deoxynucleotidyl Materials and methods. transferase (TUNEL) assay after 24, 48 and 72 h of infection with 100 MOI ofAdNull, AdWTp53 and Adp27 as previously described and according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA).7 Uninfected cells were used as controls. Briefly, TUNEL assay is based on the use ofdeoxynucleotydil transferase(TdT) showed a fainter p53 band (Figure 1a). Unfortunately, the lack ofa control protein as a proofofequal load in this experiment which catalyzes polymerization offluorescein dUTP at strand Kip1 breaks (‘nicks’) offragmentedDNA cleaved during apoptosis. makes the observation that adenovirus-mediated p27 Fluorescein labels incorporated in nucleotide polymers expression downregulates endogenous p53 only speculative. present in cells undergoing apoptosis were analyzed for flu- Further investigation will be necessary. Moreover, SUDHL-1 cells infected with Adp27 showed high level of expression of orescence by using the Epics cytometer. For each group Kip1 (mock, AdNull, and each other viral vectors) one way ANOVA p27 that was absent in the control cells (Figure 1a). These ofthe log ofthe mean intensity ( =⌺log to lin [channel number] data show that recombinant adenovirus-mediated expression × count in the channel/area) ofFITC fluorescence was was highly efficient in these cells. obtained in duplicate.7 DAPI staining cytospin Cell cycle analysis Cells were spun on slides (cytospins) at 1000 r.p.m. for 10 The effects of AdWTp53 and Adp27 on the cell cycle of min and fixed with methanol after 24, 48 and 72 h of infection SUDHL-1 cells were determined by analyzing the DNA con- with AdNull, AdWTp53 and Adp27. Uninfected cells were tent ofthe infectedcells by flow cytometry at varying times used as controls. The nuclei ofthe cells were stained with after infection. Cells kept in separated flasks were analyzed 4Ј,6-diamindino-2-phenylindole (DAPI) for fragmented DNA from 24 h to 72 h after infection. Over this time interval, the content, as previously described.9 Briefly, DAPI was applied fraction of cells in G1 showed a steady increase in SUDHL-1 for 30 min at room temperature on cytospins of infected and cells infected with AdWTp53 and Adp27, whereas no change uninfected cells. After PBS washing, slides were viewed under occurred in the uninfected cells and in the cells infected with the fluorescence microscope. AdNull (Figure 2, upper panel). Except for the cells infected with Adp27 and cells infected with AdWTp53 at 24 h, none ofthe other test populations showed a significant change over Results the time in the fraction of cells in the G2 phase (Figure 2, middle panel). Either G2 arrest or increasing level ofcell Recombinant adenoviral-mediated expression of debris related to apoptosis may cause fluctuation ofthe wtp53 and p27Kip1 in SUDHL-1 cells determined by values, as shown by the broad standard deviation in Figure 2, Western blot analysis middle panel.