(CANCER RESEARCH 55. 3716-3720. September 1. 1W] Advances in Brief Inhibition of Leukemic Cell Growth by the Protein Kinase C Activator Bryostatin 1 Correlates with the Dephosphorylation of Cyclin-dependent Kinase 21 Clement Asiedu, Joseph Biggs, Michael Lilly, and Andrew S. Kraft2 Division of Henialolofiy/Oficologv. Universitv of Alabama, iïirniinghain. Altihatna .Õ5294¡C.A., J. B., A. S. K. l ami Medical Oncolo^\. Scalile VA Medical Center. Unirersitv of Wasiiiii/itim.Scuttle.WaiHngun98108\M.LI Abstract role in regulating the growth inhibition induced by PKC-activating agents. Bryostatin 1 is a natural antineoplastic agent that activates protein The molecular basis of the growth-inhibitory effect of bryostatin 1 kinase C. Treatment of U937 human leukemic cells with bryostatin 1 is unknown. To determine its mechanism of growth inhibition and to caused a 60% reduction in cell growth, whereas another protein kinase C further examine the differences between the effects of bryostatin 1 and activator, phorbol my ristate acetate (PMA), completely inhibited U937 cell growth. Both bryostatin 1 and I'MA induced inhibition of cyclin- PMA, the activity of cdk2 in U937 cells was studied. Both compounds dependent kinase 2 (cdk2) activity. The first phase of cdk2 inhibition induced inhibition of cdk2 activity; the early phase of inhibition correlated with the transient induction of p21, a known inhibitor of cdk2. correlated with a transient induction of p21, and the later phase In contrast, the second phase of cdk2 inhibition correlated with the correlated with the dephosphorylation of cdk2 on both tyrosine and dephosphorylation of cdk2 on threonine-160, which must be phosphoryl- threonine residues. The level of cdk2 dephosphorylation on threonine- ated for cdk2 activity. The level of growth inhibition induced by these two 160 paralleled the degree of growth inhibition of human leukemic compounds correlated with the degree of cdk2 dephosphorylation as cells induced by bryostatin 1 and PMA, suggesting that cdk2 dephos follows: bryostatin 1, 60%; PMA, 100%. phorylation and inactivation may be important in the growth-inhibi tory and antitumor activity of bryostatin 1. Introduction Bryostatin 1, a naturally occurring macrocyclic lactone, is an anti- Materials and Methods cancer compound isolated from the marine bryozoan. ungula neritina Cell Culture. U937 human myeloid leukemic cells obtained from the (1). With both in vitro and in vivo anticancer activity against murine American Type Culture Collection were grown in DMEM supplemented with tumors (2), bryostatin 1 has undergone human Phase 1 testing (3). It 10% heat-inactivated bovine calf serum (GIBCO/BRL, Gaithersburg, MD) at induces the translocation of PKC1 to the plasma membrane (4), leads 37°Cin 5% CO,. Cell growth was analyzed by seeding exponentially growing to the phosphorylation of specific protein substrates (5), and eventu U937 cells at 1 X IO5 cells/ml in triplicate. Cells were left untreated or were ally induces PKC degradation (6). Although it modulates PKC in a cultured in the presence of either 200 nM bryostatin 1 or PMA. Cells were similar fashion to the tumor-promoting phorbol esters (7), it is not a counted in a Coulter counter model ZM. tumor promoter (8). As with phorbol esters, treatment of HL-60 cells Immunoprecipitation and Kinase Assays. Cells were lysed in 0.5% with bryostatin 1 induces their macrophage-like differentiation, but NP40 lysis buffer containing protease and phosphatasc inhibitors, as described unlike PMA which completely inhibits HL-60 cell growth, bryostatin (17). cdk2 immunoprecipitates were prepared from equivalent amounts of protein, and the immune complexes were assayed for histone HI kinase I only reduces the growth rate of these cells (9). activity, essentially as described (18). Treatment of U937 cells with PMA causes them to arrest in the late Northern Blotting. Isolation of total RNA and Northern blotting analysis O, phase of the cell cycle (10). Progression through the cell cycle is were carried out according to procedures described previously (19). 32P- regulated by cdks, the activation of which involves both the binding labeled p21 (2.1 kb Noll fragment from plasmid pc-WAFl-S) and a-tubulin of a cyclin partner (11) and phosphorylation and dephosphorylation of (1.5 kb Rvfl fragment) cDNAs were used as probes. specific threonine/tyrosine residues (12). For example, cdk2 is acti p21 PCR. Total RNA was isolated and subjected to reverse tnmscription- vated by the phosphorylation of threonine-160 and dephosphorylation PCR to simultaneously amplify p21 and a-tubulin sequences, using standard conditions. The four primers used were: WAF 5', 5'-CAGCTGAGCCGG- of tyrosine-15 and threonine-14 (13). Active cyclin E/cdk2 complexes GACTG; WAF 3', 5'-CGCGCTTCCAGGACTGCAGG; actin 5', 5'-CTG- are necessary for the G,-S progression of the cell cycle (11). Recent GCAAATGCACACACC, and actin 3', .V-GACAGACCCGCAAGACAA. evidence suggests that the p53-inducible protein p21 (WAF1/CIP1) is Western Blotting. Proteins from cdk2 immunoprecipitates were resolved an important downstream effector of p53-mediated G, arrest that by SDS-PAGE and transferred to Immobilon P membrane. cdk2 and anti- regulates the activity of cdk2 (14, 15). Treatment of human leukemic phosphotyrosine blots were blocked in Tris-buffered saline (TBS)/0.1% Tween cells with PMA has been shown to activate transcription of p21 by 20/5% bovine serum albumin and incubated for l h in blocking buffer con p53-indepcndent mechanisms (16), suggesting that p21 may play a taining anti-cdk2 antiserum (a gift of Dr. V. Kidd, St. Jude Children's Research Hospital) at a dilution of 1:500, and the 4G10 antiphosphotyrosine antibody (UBI, Lake Placid, NY) at a dilution of 1:1000, respectively. After washing Received 6/27/95; accepted 7/20/95. The costs of publication of this article were defrayed in part by the payment of page three times for 10 min each in TBS/0.5% NP40, blots were incubated for 45 charges. This article must therefore be hereby marked advertisement in accordance with min with second antibody in blocking buffer and washed. p21 blots were 18 U.S.C. Section 1734 solely to indicale this fact. 1This work was supported by N1H CA 42533 and ACS grant DHP 83 to ASK. blocked in PBS/1% nonfat Carnation milk and washed three times for 10 min each in TBS/0.5% BSA. After incubating with anti-p21 polyclonal antibody CA45672 and a grant from the Veterans Administration Research Program to ML. and ACS grant IRG-f>6-32 to JB. (Pharmingen, San Diego, CA), 1:1000 dilution in PBS/1% BSA for 1 h, the - To whom requests for reprints should be addressed, at Division of Hematology/ membrane was washed and incubated in PBS/1% nonfat Carnation milk for 30 Oncology. University of Alabama/Birmingham. 558T Lurleen B. Wallace Tumor min. Blots were washed and then incubated with second antibody for 45 min Institute, 1824 Sixth Avenue South. Birmingham, AL 35294-3300. *The abbreviations used are: PKC', protein kinase C; PMA. phorbol myristate acetate; in PBS/1% BSA and then washed extensively. All blots were developed with cdk, cyclin-dependent kinase. Renaissance chemiluminescence reagent (NEN Dupont, Boston. MA). 3716 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. PKC ACTIVATORS INDUCE CDK2 DBPHOSPHORYIATION In Vivo 32P Labeling. Exponentially growing U937 cells were treated with cells also demonstrated inhibition of cdk2 activity (Fig. \B). PMA 200 nM bryostatin 1 for various periods of time. [12P]Orthophosphate labeling treatment of U937 cells induced a 70-80% drop in cdk2 activity and cdk2 irnmunoprecipitation were performed, as described previously within 4-8 h, followed by a total inhibition of activity by 24-72 h. (17, 20). These data suggest that the degree of bryostatin 1 and PMA inhibition of cdk2 activity correlates with the level of growth inhibition induced Results by these agents. To evaluate the effect of bryostatin 1 on human leukemic cell Recent studies demonstrate that cdk2 activity is inhibited by p21 growth, U937 cells were cultured in the presence of either bryostatin (14). PMA treatment of U937 cells stimulates an increase in p21, 1 (200 nM) or PMA (200 nM), and cell numbers were quantitated for suggesting that p21 may be important in the regulation of the cell 5 consecutive days. Although PMA completely inhibited U937 cell cycle in response to PMA (16). To determine whether bryostatin 1 growth, bryostatin 1 caused only a 60% reduction in cell growth up to also induced p21, U937 cells were treated for varying periods of time, day 5 (Fig. 1/4). Even though bryostatin 1 was growth inhibitory, and p21 mRNA expression was analyzed by Northern blot analysis. U937 cells continued to divide slowly in the presence of this com Bryostatin 1 caused a dramatic transient induction of p21 mRNA (Fig. pound. Similar growth results with bryostatin 1 have been reported 2A). p21 mRNA expression peaked at 4 h and declined thereafter until previously (9). no message was detectable by 24-48 h (in contrast to tubulin control), The transit of cells through the cell cycle is controlled by cyclin/cdk (Fig. 2A). complexes. Treatment of U937 cells with PMA causes them to arrest Since bryostatin 1 is an anticancer agent that potentially will be in the late G, phase of the cell cycle (10). Because active cdk2 is used to treat patients with solid tumors and leukemias, it was of important in regulating the G,-S transition, the levels of cdk2 activity interest to determine whether bryostatin 1 also induced p21 in normal were evaluated after bryostatin 1 or PMA treatment. cdk2 was immu- bone marrow and fresh leukemic cells. Using reverse transcribed noprecipitated from treated U937 cells, and cdk2 activity was meas cDNA and PCR, bryostatin 1 is shown to induce p21 mRNA in normal ured in vitro using histone HI as a substrate.