[CANCERRESEARCH54,4879-4884, September15. 19941 ABSTRACT To elucidate the regulation of protein phosphatases types 1 (PP1) and 2A (PP2A) during all-trans retinoic acid (ATRA)-induced granulocytic differentiation of HL-60 cells, the phosphatase activity, proteins, and gene expressions ofPPl and PP2A were examined. Treatment with 1 pM ATRA caused an 85% decrease in the PP2A activity In extracts from HL-60 cells, while the PP1 activity was constant. This reduction in PP2A activity appeared to parallel phenotypic and functional changes of HL-60 cells Induced by A1'RA. Western blot analysis showed that the level of PP2A catalytic subunit (PP2A-C) decreased during the course of ATRA-induced differentiation, whereas expressions of A and B (Mr 55,000) regulatory subunits of PP2A were relatively Unaltered. Expressions of PP1 catalytic subunit isozymes (PP1a, PP1'y, and PP18) were not significantly affected by ATRA treatment. Northern blot analysisrevealedthat mRNA levelsof PP2A-C@ and Aa regulatory subunits were decreased following treatment with ATRA, while levels of PP2A-Ca and B (Mr 55,000) a regulatory subunit transcripts were relatively constant. Selective down regulation of PP2A-Cf3 preceded the granulocytic maturation induced by ATRA. Ex pressions of PP2A-C isoforms and A and B regulatory subunits may be differentially modulated during ATRA-induced granulocytic differentia donofHL-60cells. INTRODUCTION ATRA3 induces granulocytic differentiation in cultured leukemic HL-60 cells (1, 2) and is clinically effective as the differentiation therapy in inducing high remission rates in patients with acute pro myelocytic leukemia (3, 4). The biological effects of ATRA appear to be mediated through a number of closely related nuclear retinoic acid receptors that possess discrete DNA-binding and retinoic acid-binding domains (5). Although the exact mechanism of the ATRA-induced granulocytic differentiation remains to be elucidated, protein phos phorylation/dephosphorylation is also thought to be a regulatory de vice eminently suited for the control of differentiation processes (6). The levels of both protein kinase C activity and the expression of its isoforms have been shown to increase during HL-60 cell differentia tion induced by dimethyl sulfoxide and ATRA (7). However, little is known concerning protein phosphatases responsible for reversing the actions of protein kinase-catalyzed phosphorylation reactions in HL-60 cell differentiation. The protein serine/threonine phosphatase catalytic subunits of mammalian cells comprise four forms which have been designated type 1 (PP1), type 2A (PP2A), type 2B (calcineurin), and type 2C (8, Received 3/16/94; accepted 7/11/94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported in part by grants for research from The Ministry of Education, Science, and Culture, Japan. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: ATRA, alh-trans-retinoic acid; PP1, protein phosphatase type 1; PP2A, protein phosphatase type 2A; PP2A-C, the catalytic subunit of PP2A; MLC, myosin light chain; OK.A,okadaic acid; NBT, nitrobluetetrazolium;cDNA, complementary DNA, SDS, sodium dodecyl sulfate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. 9). They differ in metal ion requirements and sensitivities to two heat-stable protein inhibitors, inhibitor-i and inhibitor-2 (8, 9). Sev eral isoforms of PP1 catalytic subunit, PP1a, PP1'y, and PP1@ have been cloned from rat cDNAs (10). PP1s consist of multimeric struc tures composed of a catalytic subunit complexed to several regulatory subunits (8, 9). Formation of heteromeric complexes is thought to play an important role in regulating the activity of PP1 catalytic subunit, as yet not defined in HL-60 cells. The PP2A catalytic subunit (PP2A-C, Mr 36,000) is mainly present as a holoenzyme forming a heterotrimer with A (Mr 65,000) regulatory and different B regulatory subunits; the basic form is the PP2A-C/A complex, and the B subunit is associated with the dimer through A subunit (9, 11, 12). The A and B regulatory subunits were shown to modulate the phosphatase activity of the PP2A-C in vitro. cDNA clones for the two respective isoforms of PP2A-C and A subunit, PP2A-Ca and PP2A-C(3 and Aa and A(3, respectively, have been isolated from several animal species (13—15). Based on biochemical evidence, the B subunit of PP2A holoenzyme is comprised of several distinct families of proteins of B (M1 55,000), B' (Mr 54,000), B― (Mr 74,000), and Mr 72,000 (9). Three cDNAs for isoforms of the B (Mr 55,000) subunit (a, @3, ‘y) have been cloned from human, rabbit, rat, and yeast libraries (9, 16). A, B, and PP2A-C subunits show a high degree of sequence conservation among species (9). Although the effect of the regulatory subunits of PP2A on the substrate specificity has clearly been demonstrated in vitro (8,9), the roles of individual regulatory proteins in the regulation of metabolism, growth, differentiation, and development remain largely unknown. We reported that OKA and calyculin-A, both potent and specific inhibitors of PP1 and PP2A, augment the granulocytic differentiation of HL-60 cells induced by ATRA but not the monocytic differentia tion induced by phorbol diester (17). In addition, PP2A-C is down regulated during ATRA-induced granulocytic differentiation of HL-60 cells, whereas the PP1 catalytic subunit is unchanged (18). To determine the effects of ATRA treatment on regulation of cellular PP2A holoenzyme, we analyzed PP2A activity and expressions of the A and B regulatory subunits in addition to that of PP2A-C, following ATRA treatment. MATERIALS AND METHODS Cells, Culture Conditions, and Evaluation of Differentiation. Proce dures for the maintenance of HL-60 cells and determination of variable cell counts have been described previously (19). Cells in the logarithmic growth phase were used for experiments. The extent of differentiation by ATRA (Sigma Chemical Co., St Louis, Mo) was assayed by the ability to produce superoxide, as monitored by the reduction of NBT (Sigma) and surface antigen analysis. The ability of NBT reduction was evaluated as described elsewhere (20). Surface antigens were assessed by cytofluorometry in a fluorescence activated cell sorter scan (Becton Dickinson, Mountain View, CA), using monochonal antibodies, including CD! lb (0KM!; Ortho Diagnostic System Inc., Raritan, NJ), CDllc (LeuM5; Becton Dickinson), and CD54 (anti-ICAM; Cosmo Bio Co., Tokyo, Japan). 4879 Expression of the Catalytic and Regulatory Subunits of Protein Phosphatase Type 2A May Be Differentially Modulated during Retinoic Acid-induced Granulocytic Differentiation of HL-60 Cells1 Masakatsu Nishikawa,2 Serdar B. Omay, Hideki Toyoda, Isao Tawara, Hiroshi Shima, Minako Nagao, Brian A. Hemmings, Marc C. Mumby, and KatsUmi Deguchi The 2nd Department of internal Medicine. Mie University School of Medicine, E4obashi, Tsu, Mie 514 Japan (M. N.. S. B. 0., H. T., i. T., K. DI; Carcinogenesis Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, 104 Japan (H. S., M. N.]; Friedrich Miescher-institut, CH-41X12, Base!, Switzerland (B. A. H.]; and Department of Pharmaco!ogy, University of Texas. Southwestern Medica! Center, Da!!as, Texas 75235-9041 (M. C. M.] Research. on November 27, 2021. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from