Leukemia (1998) 12, 1944–1950  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu p21Cip1/WAF1 is important for differentiation and survival of U937 cells M Asada1, T Yamada1, K Fukumuro2,3 and S Mizutani1

1Department of Virology, The National Children’s Medical Research Center; and 2Department of Pharmacotherapeutics, Tokyo Science University, Tokyo, Japan

Vitamin D3 (VD3) induces monocytic differentiation of U937 and maintenance of survival of differentiated cells in mam- cells. Induction of p21Cip1/WAF1(p21) and subsequent G0/G1 mals. cell-cycle arrest are required in this process. Using a system The role of p21 in the developmental process has been of inducible expression of ectopic p21, we demonstrated the 13–15 important role of p21 in the induction of monocytic differen- investigated in p21-knock out mice. However, the results tiation in U937 cells. Prior induction of antisense-p21 of these studies are difficult to interpret due to the redundancy expression significantly suppressed p21 expression, and of molecules that have similar functions to those of p21. resulted in inhibition of VD3-induced U937 differentiation. In the present study, we examined the role of p21 in the Moreover, induction of expression of antisense-p21 in VD3-dif- induction of differentiation and survival of differentiated U937 ferentiated U937 cells resulted in apoptosis of the cells. This was associated with activation of Cdc2 and caspase-3 like pro- cells. Our findings may provide a useful model of the molecu- tease. Our results suggest that p21 is required for the initiation lar mechanisms of cell differentiation and survival at least with of the early steps of differentiation as well as survival of differ- regard to the development of monocytes in hematopoietic entiated cells. organs. Keywords: cell cycle; differentiation; apoptosis; p21Cip1/WAF1; cyclin-dependent-kinase inhibitors Materials and methods

Introduction Plasmid construction and transfection

The control of cell proliferation and terminal differentiation is The p21Cip1/WAF1(p21) cDNA was obtained by PCR ampli- dependent on the function of growth factors, oncogenes and fication using a TPA-treated U937 cDNA as template and a cell-cycle regulators. Terminal differentiation is, in principle, set of primers 5Ј-GGAAGCTTCCTGCCGAAGTCAGTTCCT- associated with an irreversible withdrawal from the cell cycle, TGTGGA-3Ј and 5Ј-CCAAGCTTCCTGTGGGCGGATTA- and the molecular mechanisms involved in proliferation are GGGCTT-3Ј. The p21 cDNA was cloned as a HindIII fragment repressed in differentiation.1 Recently, a family of negative into the zinc-inducible pMT-CB6+ eukaryotic expression vec- regulators of cell-cycle control, the cyclin-dependent-kinase tor, which contains the cDNA under the control of the sheep inhibitors (CKIs), has been isolated and the relationship metallothionein promoter, and the neomycin resistance gene between cell-cycle arrest and terminal differentiation pro- driven by the SV40 early promoter. For the antisense p21 vec- cesses is now a matter of great interest to several investigators. tor, the p21 N-terminal region was amplified using a set of CKIs bind Cdks or cyclin–Cdks complexes, and block their primers 5Ј-GGAAGCTTCCTGCCGAAGTCAGTTCCTTGT- activity, resulting in the inhibition of cell-cycle progression. GGA-3Ј and 5Ј-CCAAGCTTTTATACCTTTCTCTTCTTTTTTG- Several members of the CKI family have been identified, GCTTGGGCAGGCCAAGGCCCCGCACAC-3Ј. The nucleo- which are classified into two major groups, namely the tide sequence and orientation of the inserted DNA was con- p21Cip1/WAF1(p21) and p16INK4A groups. These CKIs exhi- firmed by sequencing. The vector DNAs were electroporated bit certain differences in the mechanism of action and in into U937 cells, which were selected in media containing specificity for Cdk targets.2 G418 (2 mg/ml) for 3 weeks. Neomycin-resistant clones were 3 ␮ p21 can be transcriptionally activated by wild-type p53 isolated and then tested in the presence of 120 M ZnSO4 for and plays an essential role in the p53-dependent G1 check- inducibility of each p21 protein by immunoblot analysis using point.4 p21 gene transcription is regulated by multiple cis-act- antibody against p21. Antisense-p21 expressing neomycin- ing elements that are responsive to several inducing signals resistant clones were tested for inducibility of p21 antisense other than p53.5–9 The induction of p21 expression has also mRNA by Northern blot. been shown in several systems inducing in vitro differen- tiation.6,10,11 The fat-soluble vitamin D3 metabolite, 1,25- dihydroxyvitamin D3 (VD3) is one such signal. VD3 contrib- Cell culture, antibodies and reagents utes to cell-cycle arrest and leads to the monocyte/ macrophage differentiation of U937 cells, by induction of Cells were cultured in RPMI 1640 (GIBCO BRL, Gaithersburg, p21.7 A sustained expression of p21 is also essential for the MD, USA) supplemented with 10% heat-inactivated fetal bov- 12 ° survival of differentiating neuroblastoma cells. Thus, p21 ine serum (FBS; GIBCO-BRL) under 5% CO2 at 37 C. Anti- appears to play multiple roles in the differentiation-induction p21 monoclonal antibody (mAb, C24420), anti-p27 mAb (K25020), and anti-Cdc2 mAb (C12720) were purchased from Transduction Laboratories (KY, USA). Polyclonal anti-cyclin A antibody (06–138) and polyclonal anti-cyclin E antibody (06– Correspondence: S Mizutani, Department of Virology, The National 134) were purchased from Upstate Biotechnology (NY, USA). Children’s Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Anti-cyclin B mAb (14551A), anti-Rb mAb (14001A), and Tokyo, 154 Japan; fax: 81 3419 4757 3Present address: Division of Hospital Pharmacy, Tokyo Women’s polyclonal anti-Cdk2 antibody (14771A) were purchased from Medical College, 8-1 Kawado-cho, Shinjuku-ku, Tokyo, Japan Pharmingen (San Diego, CA, USA). Received 26 May 1998; accepted 17 August 1998 1,25-dihydroxyvitamin D3 was obtained from Dulphar p21Cip1/WAF1 in differentiation and survival of U937 M Asada et al 1945 (Amsterdam, The Netherlands) and C2-ceramide was pur- Nitroblue tetrazolium (NBT) reduction test chased from Wako (Tokyo, Japan). TNF␣ was a kind gift from Dai-Nippon Pharmaceutical (Tokyo, Japan). Ac-DEVD-MCA, Differentiation of U937/CB6-p21 was assessed by the ability Ac-YVAD-MCA and AMC, reference compounds used for of U937 cells to produce superoxide. Production of superox- analysis with peptidyl-MCAs, were purchased from Peptide ide was measured by the NBT reduction test, using the proto- Institute (Osaka, Japan). col described previously by Collins et al.16

Metabolic labeling, immunoprecipitation and in vitro Northern blot, RT-PCR and Western blot analyses kinase assays

Total RNA was extracted from cells by a standard AGPC In vivo labeling of proteins with 35S-methionine (TRAN35S- method. For Northern blot analysis, mRNA was purified from LABEL, ICN Pharmaceuticals, CA, USA) was performed ␮ total RNA using 40 l of Dynabeads oligo d(T)25 (Nihon according to the standard technique. Cells were suspended at Dynal, Tokyo) and electrophoresced in 1% Seakem agarose 3 × 106/ml in Tween 20 lysis buffer (50 mM HEPES pH 7.5, gel (FMC, Rockland, ME, USA). RT-PCR was performed using 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, I mM DTT, 0.1% the following sets of primers: 5Ј-GGAAGCTTCCTG- Tween 20) containing protease inhibitors, and quickly son- CCGAAGTCAGTTCCTTGTGGA-3Ј and 5Ј-CCAAGCTTC- icated on ice. Immune complexes were immunoprecipitated CTGTGGGCGGATTAGGGCTT-3Ј for p21, 5Ј-GGAAGCTTG- from clarified cell lysates using protein A/G agarose (Protein GCGGTCGTGCAGACCCGGG-3Ј and 5Ј-CCAAGCTTT- A/G plus agarose, sc2003; Santa Cruz, Santa Cruz, CA, USA), TACTCGGGCAAGCTGCCCTTCT-3Ј for p27, and 5Ј-ACC- precoated with an antibody to cyclin or Cdks. For in vitro CCCACTGAAAAAGATGA-3Ј and 5Ј-ATCTTCAAACCTCCA- kinase assay, agarose was resuspended and incubated for 30 TGATG-3Ј for ␤2MG. p21, p27 and ␤2MG cDNA probes min at 30°Cin20␮l of kinase buffer (50 mM HEPES pH 7.3,

were radiolabeled using PCR. Cells were suspended in three- 10 mM MgCl2,5mM MnCl2,1mM DTT) supplemented with detergent lysis buffer (150 mM NaCl, 1.0% NP-40, 0.1% SDS, 50 ␮M ATP, 10 ␮Ci of ␥-32P-ATP (Amersham Japan) and 0.5 1.0% sodium deoxycholate, 5 mM EDTA, 10 mM Tris, pH 7.4) ␮g of histone H1 (Sigma, Tokyo, Japan). containing protease inhibitors, and quickly sonicated on ice. The protein concentration was measured using a commercial DC Protein Assay (BioRad, Tokyo, Japan). Finally, 30 ␮gof Analysis of caspase activity soluble total cellular protein was electrophoresed in a Multi- Gel 15/25 (Daiichi Pure Chemicals, Tokyo, Japan), and trans- Activities of CPP32- and ICE-like cystein proteases were ana- ferred to a PVDF membrane (Amersham Japan, Tokyo, Japan). lyzed using Ac-DEVD-MCA or Ac-YVAD-MCA as a substrate, Binding of the primary antibody was detected using a com- respectively. Cells were suspended in a lysis buffer (0.5% non- mercial ECL kit (Amersham Japan). idet P-40, 0.5 mM EDTA, 150 mM NaCl, 50 mM Tris, pH 7.5) and kept on ice for 30 min. After centrifugation, the super- natants were collected. Enzyme reactions were performed in a reaction buffer (10 mM HEPES, 0.1 M NaCl, 5 mM DTT) sup- Flow cytometric analysis plemented with 100 ␮M of Ac-DEVD-AMC or Ac-YVAD-AMC at 37°C for 2 h. The fluorescence of released AMC was meas- Flow cytometric studies of cell-surface antigens were perfor- ured by a fluorescence spectrophotometer F-2000 (Hitachi, med according to standard techniques, using mAbs directed Japan) with an excitation and wavelengths of 365 and 450 against monocyte-specific antigens, CD14 and CD11b. Anti- nm, respectively. One unit was defined as the amount of body binding was detected with a fluorescent rabbit anti- enzyme that liberated 1 nmol of AMC during a period of 2 h. mouse antibody (Wako). Flow cytometry was performed on a Becton Dickinson FACSort, and 10 000 events were analyzed. For DNA staining analysis, U937 cells were washed with Results ice-cold PBS (Mg+2- and Ca+2-free, 1% bovine serum albumin) and permeabilized with 70% ethanol at −20†C for 20 min. Ectopic p21 expression induces monocytic Cells were washed twice with PBS, then the cell pellets were differentiation of U937 cells resuspended in 1 × 106/ml of 1 × PBS, treated with RNase A (50 ␮g/ml), and stained with propidium iodide (5 ␮g/ml) at In a stable U937 transfectant containing p21Cip1/WAF1(p21) 37°C for 30 min. Flow cytometry was performed on a Becton cDNA (U937/CB6-p21) as part of the heavy metal inducible + 17 ␮ Dickinson FACSort (CA, USA), and 10 000 events were ana- vector pMT-CB6 , the addition of zinc ions (120 M ZnSO4) lyzed. For cell-cycle analysis, data were processed using the to the culture medium induced p21 mRNA within 2 h (Figure ModFit LT software (Verity Software House, ME, USA). 1a). p21 protein was the first to become detectable around 6 For analysis of apoptosis, the number of U937 cells with h after zinc induction as determined by Western blot analysis reduced DNA contents was determined first and the data were (Figure 1b). Constitutional p27Kip1(p27) expression was expressed as percentage of apoptotic cells relative to the total observed and no increase was seen during the induction of number of cells. Apoptosis was also analyzed by TUNEL differentiation in U937/CB6-p21 cells (Figure 1b). Cell-cycle assay, which was performed using the Apoptag kit (Oncor, analysis showed that the expression of ectopic p21 resulted Gaithersburg, MD, USA). DNA ends were tagged with digoxy- in G0/G1 arrest in U937/CB6-p21 cells, while U937 transfec- genin-dUTP using terminal deoxynucleotidyl transferase. tants with vector alone (U937-mock) showed no change in Apoptotic cells (which underwent DNA fragmentation) were cell-cycle distribution (Figure 1c). then stained with anti-digoxigenin FITC. FITC positive cells We also analyzed the expression of CD14 and CD11b,18,19 were analyzed on a Becton Dickinson FACSort. both of which are well known markers of monocyte/ p21Cip1/WAF1 in differentiation and survival of U937 M Asada et al 1946

Figure 1 Expression of p21 induces differentiation of U937/CB6-p21. (a) Northern blot analysis of mRNA isolated from U937/CB6-p21 cells ␮ at 0 h to 3 days following addition of 120 m of ZnSO4. Northern blots were hybridized with radio-labelled p21 or actin cDNA probe. Actin mRNA is shown as a control for variations in loading and transfer. (b) Western blot analysis of p21 and p27 proteins in zinc-induced U937/CB6- p21 cells. Whole cell lysates isolated from cells that were cultured in the presence of zinc for 0 h to 3 days were resolved by SDS-PAGE, and p21 or p27 proteins were detected by Western blot analysis using antibodies directed against each protein. (c) Ectopic p21 expression induces cell-cycle arrest at the G0/G1 phase. Cell-cycle distribution of U937-mock (left panel) and U937/CB6-p21 cells (right panel) cultured for 3 days ␮ in the absence (A, B) or presence (C, D) of 120 m of ZnSO4. The DNA content in the cells was analyzed by propidium iodide staining. Data were processed using the ModFit LT (Verity Software House). The percentage of cells at G0/G1, S and G2/M is indicated. Data shown are representative of at least three independent experiments with similar features. (d) FACS profiles of monocyte/macrophage markers determined by CD11b and CD14 antigens. U937-mock (Mock) and U937/CB6-p21 cells cultured for 3 days with zinc (p21), and U937 cells cultured for 3 days in the presence of VD3 (VD3) are shown. Solid line represents staining pattern with anti-CD11b or CD14 antibody and FITC-conjugated second antibody. Broken line represents the background staining with control mouse IgG and FITC-conjugated second antibody. Data shown are representative of at least five independent experiments with similar features. (e) U937/CB6-p21 cells showed a positive reaction in NBT test. U937-mock (A) and U937/CB6-p21 (B) cells cultured for 3 days with zinc are shown.

macrophage differentiation, in zinc-treated U937/CB6-p21. FACS analyses showed that U937/CB6-p21 cells, but not mock transfectants, expressed CD11b and CD14 3 days after the addition of zinc at a concentration of 120 ␮M (Figure 1d). The mean fluorescence intensities of these markers are shown Table 1 Effect of exogenous p21 expression on surface marker in Table 1. Three days after the addition of zinc, U937/CB6- expression on U937 cells p21 cells showed a positive reaction in NBT reduction tests (Figure 1e), indicating that the expression of exogenous p21 Control IgG CD11b CD14 induces monocytic differentiation of U937 cells to a level equivalent to that obtained by treatment with VD3. Mock 10.7 ± 1.21 12.46 ± 1.47 18.02 ± 1.53 p21 13.67 ± 1.49 63.2 ± 3.67 287.38 ± 23.46 Cell-cycle specific proteins and their kinase activities VD3 11.36 ± 1.36 57.88 ± 3.24 56.15 ± 3.59 in U937 cells induced to differentiate by exogenous p21 U937-mock (Mock) and U937/CB6-p21(p21) cells cultured for 3 days with zinc, and U937 cells cultured for 3 days in the presence Changes in the expression level of cell-cycle molecules and of VD3 (VD3) were assayed for surface markers CD11b and CD14. their kinase activities were studied during p21-mediated dif- The mean fluorescence intensities ± s.d. are indicated. p21Cip1/WAF1 in differentiation and survival of U937 M Asada et al 1947 ferentiation of U937 cells. Cyclin E-, A-, and B-associated kin- sense and the other which is not, were seen. These findings ase activities were evaluated using histone H1 as a substrate. may suggest that the expression induction of antisense p21was U937/CB6-p21 cells that had undergone differentiation by the not sufficient to neutralize VD3-induced sense p21 mRNA in addition of zinc showed a loss of cyclin E-, A-, and B-associa- some populations of U937/CB6-ASp21 clone. We also ana- ted kinase activities, while these activities were maintained in lyzed p21 mRNA expression by RT-PCR 2 days after the undifferentiated U937/CB6-p21 (Figure 2a). Immunoprecipit- addition of VD3 to U937/CB6-ASp21 cells. At the point of 20 ation analyses of metabolically labeled cell lysates showed cycles of amplification no p21 specific signal was identified that differentiated U937/CB6-p21 cells expressed cyclin E at (Figure 3c), though by increasing the amplification cycles p21 a level similar to that of undifferentiated cells, whereas the signal became recognizable (data not shown). These findings expression levels of cyclin A and B decreased markedly in suggest antisense p21 N-terminal sequence significantly differentiated U937/CB6-p21 cells (Figure 2b). Immunoprecip- counteracted the expression of p21 mRNA, while p27 mRNA itation–Western analysis of Cdk2 showed that the inactive expression remained unaffected (Figure 3b). Western blot form of Cdk2 predominates among differentiated U937/CB6- analysis of U937/CB6-ASp21 cells cultured in the presence of p21 cells whereas the faster migrating, active form prevailed zinc and treated with VD3 for 2 days showed a significant in undifferentiated cells (Figure 2c). These data indicate that reduction of p21 protein expression (Figure 3c). These results the cell-cycle arrested during the late G1 to early S phase. indicate that antisense p21 N-terminal sequence can selec- Sequential Western blot analysis showed appearance of the tively inhibit the expression of p21, and suggest that p21 is hypophosphorylated form of Rb 7 hours after zinc induction, one of the important mediators of monocytic differentiation in suggesting that changes in cell-cycle kinetics parallel the level the VD3-induced differentiation system of U937 cells. of p21 induction in U937/CB6-p21 cells (Figures 1b and 2d).

Expression of p21 enhances survival of differentiated p21 is an important mediator of monocytic U937 cells differentiation in U937 cells Since p21 expression has been suggested to support the sur- To confirm the specific effects of p21 in our system, vival of several types of differentiating cells,12,20 we examined U937/CB6-ASp21 cells were established by transducing the whether p21 was also capable of promoting the survival of pMT-CB6+ vector with p21 N-terminal sequence (aa 1–75) in differentiated cells of the hematopoietic system. To test this the antisense orientation. Northern blot analysis of U937/CB6- possibility, the expression of p21-antisense gene, whose speci- ASp21 cells cultivated for 1 day in the presence of zinc (120 ficity has been confirmed earlier by the differentiation inhi- ␮ M ZnSO4) showed a smaller size mRNA corresponding to bition assay (Figure 3), was induced by the addition of zinc the N-teminal half of p21, indicating expression of antisense to cultures of U937/CB6-ASp21 cells that had already differ- mRNA is induced by this regimen (Figure 3b). Induction of entiated by a 3-day cultivation with VD3. After a 2-day culture expression of p21 antisense mRNA 1 day before treatment in media containing zinc ions, a significant increase in the with VD3 inhibited CD11b expression in U937/CB6-ASp21 percentage of apoptotic U937-ASp21 cells was observed as as determined 3 days after the addition of VD3 (Figure 3a). demonstrated by increased numbers of cells with subdiploid However, the inhibition of differentiation was incomplete, and DNA contents in PI-stained cells (Figure 4a). Apoptotic death two populations of cells, one which is effected by the anti- of these cells was confirmed by using the TUNEL assay (Figure

Figure 2 Cell-cycle related molecules and their activities in U937/CB6-p21 cells. (a) Cyclin E-, A- and B-associated kinase activities. U937/CB6-p21 cells were cultured for 3 days in the absence (−) or presence (+) of zinc. In vitro kinase assays were performed for cyclin E, A, or B immune complexes using histone H1 as a substrate. (b) Cyclin E, A, and B expression. U937/CB6-p21 cells cultured for 3 days with or without zinc were metabolically labeled with 35S-methionine. Each cyclin was quantitatively immuno-precipitated from 3 × 106 cells and the resulting immune complexes were subjected to SDS-PAGE. (c) Active and inactive forms of Cdk2. U937/CB6-p21 cells were cultured with or without zinc for 3 days. Cdk2 was immunoprecipitated from 3 × 106 cells and the resulting immune complexes were subjected to Western blot analysis using anti-Cdk2 antibody. Arrow indicates a marker with a molecular mass of 32-kDa. (d) Time course immunoblot analysis of hyper- and hypophosphorylated Rb. Whole cell lysates isolated from U937/CB6-p21 cells cultured in the presence of zinc for the indicated time periods were resolved by SDS-PAGE, and pRb protein levels were measured by Western blot analysis using antibodies directed against Rb. hyper: hyperphosphorylated form of Rb; hypo: hypophosphorylated form of Rb. p21Cip1/WAF1 in differentiation and survival of U937 M Asada et al 1948

Figure 3 Prior expression of antisense-p21 inhibits VD3-mediated differentiation in U937 cells. (a) U937-mock (Mock) and U937/CB6-ASp21 (ASp21) cells were cultured for 3 days in the presence of VD3 at the indicated concentrations with (——) or without (. . . .) prior addition of zinc for 1 day, and analyzed for CD11b by FACS. Data shown are representative of at least three independent experiments with similar features. (b) Northern blot as hybridized with radio-labelled p21 cDNA probe for the induction of sense or antisense p21 mRNA expression by zinc. ␮ U937/CB6-p21 and U937/CB6-ASp21 cells were cultured for 1 day in the presence or absence of 120 m of ZnSO4. Exogenous ASp21 mRNA (arrow with A) ran faster than exogenous p21 mRNA (arrow with F). (c) RT-PCR analysis of p21 and p27 mRNA expression. U937-mock and − U937/CB6-ASp21 cells were cultured as in (a) with VD3 at a final concentration of 10 8 M. After 48 h, poly(A)RNA was extracted, and p21 and p27 mRNA were analyzed by RT-PCR using p21-specific and p27-specific primers. ␤-2MG mRNA was also amplified as an internal control. (d) Western blot analysis of p21 protein. Whole cell lysates from an aliquot of cells in (c) were resolved by SDS-PAGE. p21 protein was detected by Western blot analysis. It should be noted that U937/CB6-ASp21 cells cultivated in the presence VD3 and Zn showed decreased amount of p21 protein compared with those without Zn.

4b). U937-mock and U937/CB6-p21 cells differentiated with leads to apoptosis through deregulation of Cdc2 activity and VD3 but apoptosis was not induced 2 days after the addition activation of DEVD-sensitive caspase activity. of zinc (Figure 4a, b). Undifferentiated U937/CB6-ASp21 cells did not undergo apoptosis in response to induction of anti- sense-p21 (data not shown), thus ruling out a possible nonspe- Discussion cific cell-killing activity by antisense-p21 mRNA or its trans- lated product. These findings suggest that p21 may play an U937 cells are known to differentiate in response to various essential role in the survival of differentiated U937 cells. extracellular stimuli such as VD3 and TPA. To investigate the To investigate the mechanism of apoptosis induced by mechanism by which extracellular stimulation is transduced downregulation of p21, we examined changes in proapoptotic intracellularly, Liu et al7 took advantage of bypassing cell sur- signals that occur in differentiated U937 cells as a result of face-mediated processes. They reported that VD3-induced dif- antisense-p21 expression. VD3-differentiated U937/CB6- ferentiation of U937 cells was associated with upregulation of ASp21 cells showed increased Cdc2 kinase activity 24 h after several CKIs, including p21, although ectopic expression of the addition of zinc (Figure 4c), whereas the level of Cdc2 p21 alone was not as effective as VD3.7 In contrast to their expression did not change during antisense-induced apoptosis findings, the major finding of the present study was that (Figure 4c). Caspase activities were also examined at 0, 24 ectopic expression of p21 alone was sufficient to induce mon- and 48 h after antisense-p21 induction. A nine-fold increase ocytic differentiation to a level similar to that observed in in DEVD-sensitive caspase (caspase 3-like protease) activity VD3-treated cells. We also showed that VD3-induced mono- was detected 48 h after zinc treatment, but no activity of cytic differentiation was restrained by prior expression of anti- YVAD-sensitive caspase (caspase 1-like protease) was sense p21 mRNA, which selectively inhibited the expression detected at these time intervals (Figure 4d). These results sug- of p21 mRNA without an associated downregulation of p27 gest that inhibition of p21 expression in differentiated cells mRNA expression. These findings imply not only that p21 p21Cip1/WAF1 in differentiation and survival of U937 M Asada et al 1949

Figure 4 p21 supports the survival of differentiated U937 cells. (a) Antisense p21 expression in VD3-differentiated U937/CB6-ASp21 causes apoptosis. U937-mock (Mock), U937/CB6-p21 (p21), and U937-ASp21 (ASp21) cells were cultured in the presence of VD3 for 3 days. Cells were then washed and resuspended in a medium containing 120 ␮m of zinc. Cells were stained by PI 2 days later and the population of apoptotic cells identified as a subdiploid peak was analyzed by FACS. Data are representative of at least three independent experiments. (b) TUNEL assay. Cells treated with zinc as in (a) were stained by the TUNEL method and analyzed by FACS. (c) Cdc2 activities in cells undergoing apoptosis by antisense p21 expression. Cells were treated with zinc as in (a). Cdc2 immune complexes were prepared 24 h after the addition of zinc and an in vitro kinase assay was performed using histone H1 as a substrate (top). Cdc2 expression level in VD-3 differentiated U937/CB6- ASp21 cells with or without antisense-p21 expression was not different after 24 h (bottom). Whole cell lysates were resolved by SDS-PAGE, and Cdc2 protein levels were measured by Western blot analysis using antibody against Cdc2. (d) DEVD-sensitive caspase activation was detected in antisense-p21 induced apoptotic cells. Cells were treated with zinc as in (a). YVAD- and DEVD-sensitive caspase activities were determined 0, 24, and 48 h after zinc induction as described in Materials and methods. Data are representative of at least three independent experiments. expression is required in the initial step of VD3-induced mon- ible involvement of de novo induction of Cdc2 protein in the ocytic differentiation, but that it is by itself sufficient to induce upregulation of the activity of this kinase. Since the kinase monocytic differentiation. The difference between our find- activity of Cdc2-Cyclin B is positively regulated by Cdk2 kin- ings and those of Lui et al7 may be due to the systems used ase,21 Cdc2 must have been activated through the release of for the induction of expression. An inducible expression sys- Cdk2 activity caused by downregulation of p21. While further tem on a stable transfectant was used in the present study, studies are necessary in order to elucidate the mechanism of whereas a transient expression assay was used in the other Cdc2 activation, it is conceivable that inappropriate activation study. These differences illustrate the need for a careful of Cdc2 kinase might cause apoptosis of differentiated U937 interpretation of individual findings. cells. This argument is based on previous findings demonstrat- If p21 expression could lead by itself to differentiation of ing that premature activation of the serine-threonine kinase U937, what is then the role of p21 in the whole cellular Cdc2 precedes apoptotic cell death,22 and the disassembly of machinery that induces differentiation of U937? It is possible the nuclear lamins by Cdc2 activation observed in cells that the mechanisms responsible for the progression of differ- entering mitosis causes morphological changes similar to entiation program may involve p21-independent machinery. those found in cells undergoing apoptosis.23 Induction of cell cycle arrest by ectopic p21 expression may DEVD-sensitive caspase activity, which is closely involved only allow progression of such differentiation program to con- in the transmission of proapoptotic signals,24 showed approxi- tinue when already set to work. Alternatively, p21 might mately nine-fold increase during antisense-induced apoptosis directly trigger the differentiation program in U937 cells, (Figure 4d), indicating that the DEVD-sensitive caspase is also which may or may not act through activation of mechanisms involved in this process. Since p21 is known to inhibit the that halt cell-cycle progression. In this scheme, VD3-induced activation of SAPK/JNK,25 future studies should determine differentiation is exclusively dependent on such p21 activities. These findings are in disagreement with those described in whether stress-activated MAP kinase is involved in the myoblast differentiation, where the activity of p21 is thought machinery of apoptotic cell death. It is intriguing to speculate to be unnecessary for the initiation of differentiation, but that a positive feedback loop between activation of MAP kin- 26 rather for maintenance of such a process.1 ase and that of caspase, which amplifies the apoptotic Our results also showed that specific inhibition of p21 response, may somehow be involved in this process. expression in VD3-differentiated U937 cells by antisense p21 Considered together, our findings suggest that p21 plays an induction caused apoptosis of these cells. Furthermore, our important role in cell-cycle arrest, cell differentiation, and cell analysis showed upregulation of Cdc2 activity 24 h after anti- survival at least in our model using U937 cells. We are cur- sense-p21 induction, which was followed by apoptosis within rently conducting further studies to investigate whether these 48 h. The expression level of Cdc2 did not change during mechanisms are also involved in the regulation of differen- apoptosis by antisense-p21 induction, thus ruling out a poss- tiation of normal cells of the hemopoietic system. p21Cip1/WAF1 in differentiation and survival of U937 M Asada et al 1950 Acknowledgements of the tumour suppressors IRF-1 and p53 in response to DNA dam- age. Nature 1996; 382: 816–818. 10 Jiang H, Lin J, Su Z-Z, Collart FR, Huberman E, Fisher PB. Induc- We are grateful to Dr Fujimoto from the Department of Pathol- tion of differentiation in human promyelocytic HL-60 leukemia ogy, the National Children’s Medical Research Center, for the cells activates p21, WAF1/CIP1, expression in the absence of p53. generous gifts of anti-CD11b and anti-CD14 antibodies. We Oncogene 1994; 9: 3397–3406. also thank Drs Tsujimoto and Miyashita for the critical reading 11 Steinman RA, Hoffman B, Iro A, Guillouf C, Liebermann DA, El- of this manuscript. We thank Mrs M Komai for technical assist- Houseini ME. Induction of p21(WAF1/CIP1) during differentiation. ance and Dr Miyauchi for technical advice. This work was Oncogene 1994; 9: 3389–3396. 12 Poluha W, Poluha DK, Chang B, Crosbie NE, Schonhoff CM, Kil- supported by a Grant-in-Aid for Pediatric Research (6–5) from patrick DL, Ross AH. The cyclin-dependent kinase inhibitor the Ministry of Health and Welfare, Japan, by a Grant-in-Aid p21WAF1 is required for survival of differentiating neuroblastoma for Cancer Research from the Ministry of Health and Welfare, cells. Mol Cell Biol 1996; 16: 1335–1341. Japan, by a Grant-in-Aid from the Ministry of Health and Wel- 13 Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, fare, Japan, as part of a comprehensive 10-year strategy for Hannon GJ. Radiation-induced cell cycle arrest compromised by Cancer Control, by a Grant-in-Aid from the Ministry of Edu- p21 deficiency. 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