Comparison of the Effects of Recombinant Adenovirus-Mediated

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 application 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 adenoviral 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. The cell debris causes events that are most vis- ible on the left side of the G1 peak of the cell cycle profile SUDHL-1 cells expressed high level ofwtp53 and p27 Kip1 after (not shown).18 The events may also extend into the lowest and 24 h ofinfectionwith AdWTp53 and Adp27, as defined by broadest G2-S compartment of the histograms. This affects the Western blot analysis (Figure 1a and b). As previously shown, calculations ofthe G2 and S phases. The values will be higher SUDHL-1 infected with AdNull and uninfected cells (mock) than expected ifmodeling ofthe debris distribution in the his- showed some level ofendogenous expression ofp53 (Figure togram analysis is not applied, as in our case.18 The fraction 1a).7 The intensity ofthe band correspondent to the expected ofcells in the S phase steadily decreased more in cells size ofp53 was 1/3 ofthat present in cells infectedwith infected with Adp27 than in those with AdWTp53 compared AdWTp53 (Figure 1a). SUDHL-1 cells express a mutated p53 to the controls (Figure 2, lower panel). Adp27 was as efficient (unpublished data). Surprisingly cells infected with Adp27 as AdWTp53 in inducing persistent G1 arrest in SUDHL-1.

Leukemia Cell cycle and apoptosis induced by exogenous expression of wild-type p53 and p27Kip1 F Turturro et al 1227 agreement with previous observations.7 Therefore, the shift of the intensity ofFITC fluorescence in cells after24 h ofinfection with AdWTp53 compared to uninfected cells was less pronounced than that showed by cells infected with Adp27 compared to the respective controls (Figure 3a, upper panel). A higher level ofapoptosis occurred in cells infectedwith Adp27 after 24 h. On the contrary, the shift of the intensity ofFITC fluorescence compared to the controls was less pronounced in cells infected with Adp27 than in those infected with AdWTp53 at 48 and 72 h (Figure 2a, mid and lower panel). As shown in Figure 3b, brightly stained nuclei and cell debris appeared in 80% ofcells afterDAPI staining of SUDHL-1 cells 48 h after infection with AdWTp53, but not in the uninfected cells. Cells infected with Adp27 had less bright nuclei and more intact cells compared to those infected with AdWTp53 at the same time ofinfection(Figure 3b). We then compared the fraction of cells in G1 phase and the level of apoptosis detected by TUNEL analysis at different times ofinfectionin the cell populations. Whereas cells infected with AdWTp53 and Adp27 showed a persistent increase ofpercentage ofcells in G1 phase at all times of infection, they presented different levels of apoptosis (Figure 4a and b). The mean FITC fluorescence derived from duplicate histograms ofcells stained with TUNEL and analyzed by flow cytometry showed that cells infected with AdWTp53 had less apoptosis than cells infected with Adp27 at 24 h of infection (Figure 4b). The mean FITC fluorescence progressively increased in cells infected with AdWTp53 at 48 and 72 h, whereas it was less pronounced and stable in cells infected with Adp27 at the same times ofinfection(Figure 4b). These data suggested that apoptosis occurred rapidly following G1 arrest in cells after 24 h of infection with Adp27. The magni- tude ofapoptosis decreased and stabilized with persistent G1 arrest after 48 and 72 h of infection in the same cell popu- lation (Figures 3 and 4). Although apoptosis occurred later in cells infected with AdWTp53, it was more pronounced and persistent at any time after 24 h (Figures 3 and 4). This last finding was in agreement with previous observations.7

Figure 2 Cell cycle analysis. SUDHL-1 cells were stained with PI and analyzed by flow cytometry at different times of infection with 100 MOI ofAdNull, AdWTp53 or Adp27. Uninfectedcells (mock) Dose–effect relationship between MOI of viral were used as controls. Histograms ofthe percentage ofcells in G1 particles, cell cycle and apoptosis induced by Adp27 (upper panel), G2 (mid panel) and S (lower panel) from duplicate in SUDHL-1 cells experiments expressed as mean ± s.d. are shown. We analyzed SUDHL-1 cells for DNA content (cell cycle) 48 h after infection with 50 and 100 MOI of Adp27, respectively. Analysis of the apoptosis induced by AdWTp53 and The percentage ofcells in G1 phase infectedwith 50 MOI of Adp27 Adp27 (87.7%) was similar to those infected with 100 MOI ofAdp27 (86.5%), as shown in Figure 5, right side. The per- To assess the effects of the persistent G1 arrest induced by centage ofcells in S phase was reduced in both cell popu- AdWTp53 and Adp27 in SUDHL-1, we measured the extent lations compared to the controls (Figure 5). This clearly of apoptosis occurring at 24, 48 and 72 h after infection as showed that the effect of adenoviral-mediated expression of a consequence ofthe overexpression ofwtp53 and p27 Kip1, p27Kip1 on cell cycle was not related to the dose ofviral respectively. Aliquots of cells from different times of infection particles. were stained with TUNEL and DAPI. TUNEL-stained cells We then analyzed the level ofexpression ofp27 Kip1 induced were analyzed by flow cytometry, whereas DAPI-stained cells by the recombinant adenovirus by Western blot analysis in were examined with fluorescence microscope. cells after 24 h of infection. As shown in Figure 6a, cells The shiftofthe histogram ofthe intensity ofFITC fluor- infected with 100 MOI of Adp27 showed a band within the escence in cells infected with AdWTp53 or Adp27 was com- expected kDa ofp27 Kip1 that was three-fold more intense than pared to uninfected cells at different times of infection. We the one present in cells infected with 50 MOI of Adp27. These decided to use uninfected cells because the histogram of the data demonstrated that 50 MOI ofrecombinant adenovirus intensity ofthe FITC fluorescence ofcells infectedwith AdNull expressed enough protein to induce G1 arrest in 88% of did not show any shift compared to the uninfected cells (not cells infected. shown). These data were consistent with lack ofaspecific tox- We finally assessed the level ofapoptosis in cells infected icity related to the recombinant adenoviral vector and was in with different MOI of Adp27 after 48 h of infection by TUNEL

Leukemia Cell cycle and apoptosis induced by exogenous expression of wild-type p53 and p27Kip1 F Turturro et al 1228 a

Figure 4 Comparison ofG1 and TUNEL data. (a) Mean ofthe percentage of cells in G1 from duplicate derived from data in the upper panel ofFigure 2. (b) Mean FITC ﬂuorescence intensity fromduplicate histograms analyzed by ﬂow cytometry and shown in Figure 3a. This was obtained by one way ANOVA ofthe log ofthe mean intensity as described in Materials and methods.

Figure 3 (a) Flow cytometry analysis ofapoptosis in SUDHL-1 assay. Cells infected with 100 MOI of Adp27 showed a more cells stained with TUNEL assay after 24, 48 and 72 h of infection with pronounced shift of the FITC fluorescence histogram com- 100 MOI ofAdNull, AdWTp53 or Adp27. Histograms ofthe FITC pared to the control by flow cytometry analysis than those fluorescence from each sample (white) with respective matched unin- infected with 50 MOI (Figure 6b). This was consistent with fected control (black) are shown as most representative of duplicate experiments. Deoxynucleotidyl transferase (TdT) catalyzes polymeriz- more apoptosis occurring in cells expressing higher levels of ation offluorescein dUTP at strand breaks (‘nicks’) offragmentedDNA p27Kip1. cleaved during apoptosis. Fluorescein labels incorporated in nucleotide polymers present in cells undergoing apoptosis were analyzed for fluorescence by using the Epics cytometer. The FITC fluorescence Discussion intensity of the histogram derived from cells infected with AdNull did not show any shift compared with the histogram derived from uninfected cells (mock). Therefore AdNull-histogram is not shown due to Previous studies have shown that adenoviral vector expressing overlap with the one derived from uninfected cells. (b) DAPI staining p27Kip1 caused G1 arrest followed by apoptosis.8–10 Some ofthe nuclei ofSUDHL-1 cells. Cells were spun on slides, stained investigators have compared the efficiency of several adenovi- with DAPI and viewed with the fluorescence microscope after 48 h ral vectors expressing cyclin-kinase inhibitors and tumor sup- ofinfectionwith 100 MOI ofAdNull, AdWTp53, or Adp27. The inten- pressor wtp53 in cell lines derived from epithelial tumors.10 sity ofthe fluorescence ofthe nucleus stained with DAPI is correlated Apoptosis was analyzed in HELA cells after 4 days of infection with the content of fragmented DNA. Cells infected with AdNull (not shown) did not show any difference in intensity of fluorescence com- with adenoviral vectors expressing p16, p18, p19, p21, p27 pared with uninfected cells (mock). Therefore uninfected cells are and p53. Cell cycle was analyzed in A549 and MT1A2 cells shown with cells infected with AdWTp53 or Adp27 (× 200). Cells after 48 h of infection. Therefore cell cycle and apoptosis were infected with AdWTp53 present higher fluorescence intensity and compared in different cell lines and they were never analyzed increased number of fragmented cells compared to those infected in the same cell line using several recombinant adenoviral with Adp27. vectors.10 Moreover, apoptosis was never analyzed after 24 h ofinfection,the time when the pathways are activated. 10 In the current study we have analyzed, for the first time, the initial events ofcell cycle and apoptosis induced by wtp53 and p27Kip1 exogenously expressed by adenoviral vectors in the same cell line. We have also compared, for the first time,

Leukemia Cell cycle and apoptosis induced by exogenous expression of wild-type p53 and p27Kip1 F Turturro et al 1229

Figure 5 Cell cycle analysis of SUDHL-1 infected with different MOI of AdNull or Adp27. Cells were stained with PI and analyzed for DNA content after 48 h of infection with 50 MOI (upper panel) or 100 MOI (lower panel) of AdNull or Adp27. The most representative histograms and the percentage ofcells in each phase fromduplicate are shown. The histograms and percentage ofcells in each phase derived from uninfected cells (mock) similar to those derived from cells infected with AdNull are not shown. the effects induced by these proteins in a cell line derived study (Figures 5 and 6). The excess ofp27 Kip1 in the cell may from a human hematopoietic malignancy. A recent study has also create an imbalance ofthe stoichiometric ratio to the shown that cleavage ofp21 Waf1 and p27Kip1 by CPP32 CDK/cyclin complexes, thus interfering with the nuclear local- (caspase 3), a member ofthe caspase cascade, mediates ization ofthe complexes. Furthermore, this event may ulti- apoptosis in endothelial cells through activation ofcyclin- mately hamper the function that low levels of inhibitors exert dependent kinase 2 (CDK2).19 This study supports our ﬁndings in stabilizing the complexes and enhancing cell cycle pro- and leads to the hypothesis that p27Kip1 expressed by the gression.22 This proposed model is undergoing further investi- adenoviral vector may bind to the cyclin A/E-CDK2 complex gation. Apoptosis occurring in SUDHL-1 cells infected with in SUDHL-1. The binding may expose a site ofthe protein AdWTp53 lags behind cells infected with Adp27 after 24 h of that is cleaved by CPP32- and/or CPP32-like caspase. The infection. As previously shown, adenovirus-mediated cleavage reduces the association ofthe inhibitor to the expression ofwtp53 induces p21 Waf1 in SUDHL-1 cells that cyclinA/E-CDK2 complex with rapid induction ofCDK2 lack a functional p53.7 We demonstrated that p21Waf1 activity. Caspase and CDK2 activation may be responsible for expression is induced after 18 h of infection with AdWTp53 the rapid onset of apoptosis observed 24 h after infection with in SUDHL-1 cells.7 The expression ofp21 Waf1 triggers G1 Adp27. It has been shown that the complex cyclin E-CDK2 arrest and consequently apoptosis.7 Apoptosis induced by directly phosphorylates p27Kip1 at the G1/S transition.20,21 This p21Waf1 is progressive and steadily increases over time in event targets p27Kip1 for the ubiquitin-dependent proteolysis.20 SUDHL-1 compared to that induced by p27Kip1 (Figures 3 We postulate that exogenously expressed p27Kip1 in SUDHL- and 4). 1 triggers several pathways responsible for inducing either G1 ALCL has recently been described as a separate clinical arrest or apoptosis. The same pathways may be involved in entity that has been recognized by the REAL classiﬁcation as the regulation ofthe protein exogenously expressed. The level a distinct non-Hodgkin’s lymphoma (NHL).23 The majority of ofp27 Kip1 expressed by the recombinant adenovirus plays a ALCL is characterized by the presence ofthe t(2;5)(p23;q35) different role in regulating these pathways as shown in this chromosomal translocation that causes the fusion of the ALK

Leukemia Cell cycle and apoptosis induced by exogenous expression of wild-type p53 and p27Kip1 F Turturro et al 1230 NPM-ALK downregulates p27Kip1 in SUDHL-1, exerting an antiproliferative effect similar to BCR-ABL. Further investigation is in process to verify this hypothesis. Moreover, more recently low levels ofp27 Kip1 have been associated with increased malignancy in lymphomas.31 In conclusion, we have shown the efﬁcacy of recombinant adenovirus-mediated expression ofwtp53 and p27 Kip1 in inducing G1 arrest and apoptosis in SUDHL-1 cells. Although we have already demonstrated the efﬁcacy of adenovirus- mediated expression ofwtp53 in inducing apoptosis and necrosis in vivo in a nude mouse model ofALCL, we further postulate the potential application ofAdp27 forgene therapy ofthis lymphoma. 32

Acknowledgements

This work was supported by a grant from the Des Moines Research and Education VA Central Iowa Health Care System, Des Moines, IA, USA.

References

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