Two distinctly regulated events, priming and triggering ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 91, pp. 8428-8432, August 1994

Cell Biology

Two distinctly regulated events, priming and triggering, during retinoid-induced maturation and resistance of NB4 promyelocytic leukemia cell line (acute promyelocytic leukemia/dlferentlation/retlnoic add/signaling cross-talk)

S. RUCHAUD*, E. DuPREz*, M. C. GENDRON*, G. HOUGEt, H. G. GENIESER*, B. JASTORFF§, S. 0. DOSKELANDt, AND M. LANOTTE*1 tCell Biology Research Group, Institute of Anatomy, University of Bergen, 5000 Bergen, Norway; *Biolog Life Science Institute and §Bio-organic Chemistry

Department, University of Bremen, 28359 Bremen, Germany; and *Institut National de la Sante et Recherche Medicale, Unite 301, Centre Hayem, H6pital Saint-Louis, 75010 Paris, France

Communicated by Pierre Chambon, April 25, 1994

(9-12). Several authors (see ref. 17) suggested that a PMLRARa chimeric protein, by altering RA signals and/or modulating PML function, was responsible for the block of the differentiation program of leukemic blasts. Consistently,

In t(15;17) acute promyelocytic leukema, ABSTRACT all-trans retinoic acid (RA) induces leukemic cell maturation in vitro and remission in acute promyelocytic leukemia patients, but in vivo treatments invariably lead to relapse with resistance to RA. NB4, a maturation-inducible cell line, and NB4-RAr sublines (Ri and R2) displaying no maturation in the presence of RA have been isolated from a patient in relapse. We show that resistance to maturation is not a mere unresponsiveness to RA: rather, R1 "resistant" cells do respond to RA (1 pM) by sustained growth, become competent to undergo terminal maturation, and up-regulate CD11c/CD18 integrins. Interestingly, maturation of "resistant" cells, rendered competent by RA, can be achieved by cAMP-elevating agents (prostglandin E, isoproterenol, cholera toxn, or phosphodiesterase inhibitor) or stable agonistic cAMP analogs such as (Sp)-8-chloroadenosine cyclic 3',5'-phosphorothioate. This shows that activation of cAMP-dependent protein kinase (cA kinase) can override the RA resistance and suggests interdependent RA and cAMP sinling pathways In acute promyelocytic leukemia maturation. No such cooperation was observed in the R2 resistant cells, though their cA-kinase was functional (Rp)-8Chloroadenosine cyclic 3',5'-phosphorothioate, which by displacing endogenous cAMP inhibits the basal cA-kinase activity, decreased the response of sensitive cells to RA. This raises the possibility that cA-kinase plays a key role in the maturation also of RA-sensitive cells. Our results define two discrete steps in the maturaton process: an RA-dependent priming step that maintains proliferation while cells become competent to undergo maturation in response to retinolds and a cAMP-dependent step that triggers RA-primed cells to undergo terminal maturation. Uncoupling RA and cAMP action might cause the so-called "resistance."

PML-RARa protein and truncated RARa protein show dominant negative effects on gene expression (refs. 13-16; for review, see ref. 17). That chimeric PML-RAR protein forms heterodimers with normal RARa and retinoid X receptor a indicates that various RA-dependent responses might be altered in APL. Those might depend on the levels of expression of the normal and chimeric RARs, their heterodimeric interactions, and DNA binding properties (18). Finally, PML-RARa protein might act also as a modulator of PML function in APL (19, 20). Whatever function PML-RAR protein has, either in APL etiology or as an effector in RA-induced maturation, the above findings indicate how intricate the response of APL to retinoids must be. This complexity is underscored by clinical data. Although RA employed in vivo therapeutically (5) induces remissions, relapse with "resistance" to retinoids rapidly follows. The cause for RA resistance is not yet clearly understood (for review, see ref. 21). To investigate in vitro biological responses of APL to retinoids, we benefit from the NB4 cell line (22), a unique cell line with the t(15;17) translocation. Interestingly, we have been able to isolate (23) a maturation-resistant line (NB4RAr) from the same sample of APL marrow. The response to retinoid was clearly heterogenous: (i) the major part of the progeny of treated cells showed growth arrest and maturation, but a minor component of blast cells achieved sustained proliferation and failed to mature, and (ii) a continuous stimulation by RA was essential to maintain resistance in blast cells. These observations lead to the hypothesis that resistance in APL is generated early in response to RA, and although cells do not mature, they remain responsive to RA

Retinoids exert regulatory function in several major biological phenomena, such as cell growth and differentiation, vertebrate development, neoplasia (for review, see ref. 1). Retinoic acid is a vitamin A derivative whose effects on gene expression are mediated by at least two families of nuclear receptors, retinoic acid receptors [RARs (a, (3, 'y)] and retinoid X receptors (a, /3, y) (for reviews, see refs. 2 and 3). Since Breitman et al. (4) have found that retinoids induced acute myeloid leukemia cell differentiation, numerous studies have focused on the effects ofthis drug on leukemia. More recently (5, 6), all-trans retinoic acid (RA) was reported to be a potent maturation inducer of acute promyelocytic leukemia (APL) cells, a myeloid leukemia that shows a typical t(15;17) translocation (7, 8) fusing RARa gene and the PML gene

(23).

In this work, sensitive and resistant NB4 cell lines allowed us to dissect RA-induced maturation into two distinctly regulated events priming and triggering. We demonstrate that maturation-resistant cells respond to RA and become competent for maturation, but maturation triggering requires a cAMP-dependent event. Abbreviations: APL, acute promyelocytic leukemia; cA-kinase, cAMP-dependent protein kinase; 8-CPT-cAMP, 844-chlorophenylthio)adenosine cyclic 3',5'-monophosphate; DMSO, dimethyl sulfoxide; NBT, nitroblue tetrazolium; PG, prostaglandin; RA, alltrans retinoic acid; RAR, retinoic acid receptor; (Sp)- and (Rp)-8C1cAMP[S], (Sp)- and (Rp)-8-chloroadenosine cyclic 3',5'-phosphorothioate. 1To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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MATERIALS AND METHODS Chemicals. RA, dimethyl sulfoxide (DMSO), prostaglandin (PG) E1, PGE2, cholera toxin, phorbol 12-myristate 13acetate, 8-(4-chlorophenylthio)adenosine cyclic 3',5'-monophosphate (8-CPT-cAMP), isobutylmethylxanthine, and nitroblue tetrazolium (NBT) were purchased from Sigma. (Rp)- and (Sp)-8-Chloroadenosine cyclic 3',5'-phosphorothioate {(Rp)and (Sp)-8C1-cAMP[S] (BioLog, Life Sciences Institute, Bremen, Germany)} are analogs ofthe parent compound cAMP in which the hydrogen in position C8 of the base is replaced by chloride (Cl) and one of the two exocyclic oxygen atoms in the cyclic phosphate moiety is modified by sulfur. Equatorial thio [S] substitution (cAMP[S]) on phosphorus (P) leads to the R isomer (Rp), and axial modification yields the S compound (Sp). The Sp configuration of this analog is an agonist, whereas the analog with an Rp configuration functions as an antagonist (24, 25). Cell Cultures and Analysis of Cell Maturation. The culture of NB4 and NB4-RAr cells is detailed elsewhere (22, 23). NB4-RAr cell line is named R1 in this work since a distinct resistant cell line, R2 was derived from R1. The R2 cell line was isolated by continuous treatment of R1 cells with RA (1 ,uM) and a potent activator of cAMP-dependent protein kinase (cA-kinase), 8-CPT-cAMP (100 zM). Each resistant cell line was grown either in RA-free medium (R[-] cells) or cultured for a week in medium with 1 AM RA (R[+] cells). Cells were incubated with RA (1 pmol/liter from stock solution at 1 mol/liter in DMSO, stored at -30°C in the dark). Morphological studies were performed on smears stained with May-Grunwald-Giemsa. Cell maturation was measured by NBT reaction (22, 23, 41) and by morphological changes. cA-Kinase Studies. Regulatory subunit (RI and RII) and catalytic subunit (C) gene expression was analyzed by Northern blot hybridization. The concentration, isozyme distribution, and cAMP responsiveness of cA-kinase were determined as described (26). Flow Cytometry Analysis of Cell Surface Antigens. The expression of CD11/CD18 integrins was analyzed by indirect immunofluorescence as described (22, 23). RESULTS From the t(15;17) promyelocytic leukemia culture that generated the NB4 cell line (22), two sublines resistant to RA [R1 (23) and R2] were isolated. Maturation-resistant cells bear the t(15;17) PML-RARA gene rearrangement and express PML, RARa, and the chimeric transcript PML-RARa as found in

Proc. Natl. Acad. Sci. USA 91 (1994)

8429

the maturation-inducible NB4 cell line [shown by Northern and Southern blot analyses (23), using specific probes (16)]. This suggests that cells resistant to RA are not the result of a gross modification in structure of PML, RARA, and PMLRARA genes. R1 and R2 cells are distinct from NB4 cells with respect to

several phenotypical features (unpublished data). Briefly, NB4 cells and R1 cells treated with RA show diverging maturation patterns. R1 cells failed to mature even at RA concentrations >10 ,M. In contrast, at low doses of RA (15-150 nM), both NB4 and R1 cells respond by enhanced cell proliferation (measured by MTT assay and cytometry cell cycle analysis), which suggested that R1 cells were responsive to RA even though they were resistant to cell maturation (data not shown). Typically, R2 cells neither matured nor showed sign of growth response to retinoids (unpublished data). R1 and R2 cell features suggested two types of resistance to retinoids and two distinctly regulated events.

Membrane Receptor Agonists Trigger Maturation of R1Resistant CelLs. We hypothesized that maturation of APL cells resulted from coupled cellular signaling pathways and, therefore, looked for means of inducing maturation of RA resistant cells. Vitamin D3 and DMSO had no effect on maturation, whereas phorbol 12-myristate 13-acetate induced some monocytic-like phenotype modifications (data not shown). DMSO (0.5-1%) exhibited low synergistic activity with RA on maturation in NB4 cells, but not in resistant cells. However, higher doses (2%) induced massive apoptosis in both NB4 and resistant cells (data not shown). Agonistic stimulation of adenylate cyclase-coupled receptors, G-protein activators, phosphodiesterase inhibitor (isobutylmethylxanthine) and cAMP analogs were tested on maturationinducible and -resistant cells. Table 1 shows that the maturation blockade of Rl-resistant cells was released by activating the cAMP signal-transduction pathway. cAMP signal induced R1[+] cells to mature but clearly not NB4 or R1[-] cells. This demonstrated that a cAMP-responsive mechanism triggers R1 cell maturation, provided that a RA-permissive signal has been delivered. Neither of the two signaling pathways is sufficient to induce cell maturation per se, but their conjunction is determining. Priming and Triggering Two Distinctly Regulated Events of APL Maturation. The above data were interpreted as follows: (i) Retinoids render APL cells competent for maturation during a step that we propose to define as maturation priming. Priming is associated with a sustained cell proliferation and

Table 1. cAMP-dependent regulation of proliferation and maturation of RA-sensitive and RA-resistant NB4 cell lines P/M ratio Treatment NB4 NB4 + RA R1[-] R1[+] + RA R2[+] + RA Saline 58/28/++ 44/38/49/Cholera toxin (10 nM) 45/3/+++ 46/6/++ 36/PGE1 (10 UM) 49/17/+++ 30/20/+++ 48/PGE2 (25 uM) 57/37/++ 41/21/+ 43/Isoproterenol (50 AM) ND/ND 9/+ + 32/ND/ND/IBMX (1 mM) 15/+++ 41/28/9/++++ 21/8-CPT-cAMP (100 ALM) 45/6/++++ 51/6/++++ 49/45/(Sp)-8C1-cAMP[S] (75 ,uM) 3/++++ 40/22/++ 45/Cells were cultured with drugs at the indicated concentrations. When cells were treated with a combination of a cAMP-elevating agent and RA, RA was used at 1 pM throughout the culture. Cultures were arrested after 48 h of incubation. In some cases, cells were preincubated with 1 pLM RA for 7 days (noted by a [+] after the name of the cell line). R1[-], R1 cells preincubated in RA-free medium; R1[+], R1 cells were preincubated with 1 pM RA for 7 days before drug treatments; R2[+], R2 cells similarly preincubated with RA. Cell proliferation [mitotic index (P)] and morphological maturation (M) were estimated on smears stained with May-Grfinwald-Giemsa. Mitotic index (P) is number of mitotic figures per 1000 cells. Evaluation of cell maturation was based on the classical morphological criteria (-, myeloblastic cells; +, myeloblasts/promyelocytes and 10%6 polynuclear cells; + +, myelocytes/metamyelocytes and 25% polynuclear cells; + + +, 50% of polynuclear cells; + + + +, 60-99% neutrophilic polynuclear cells). ND, no data; IBMX, isobutylmethylxanthine.

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using membrane expression of adhesion receptor integrins and functional maturation in response to cA-kinase activation. A tight interdependence between the RA and cAMP systems is suggested by the following observations. (i) R2 cells lack completely RA induction of a number of genes (unpublished data) induced in NB4 cells, like the maturation markers CD18/CD11c integrins (for review, see ref. 28) (Fig. 2). Indeed, we found that NB4, R1, and R2 cells expressed low levels of CDiic and CD18. RA strongly up-regulated the CD18 marker in both NB4 and R1 cells, confming that RA signaling was operative in R1 cells. In contrast, no variation was observed in R2 cells (Fig. 2A). RA also induced a strong expression of CDlic in NB4 cells and a lower but significant increase in R1 cells, while it failed to increase this marker in R2 cells (Fig. 2B). Interestingly, RA and cAMP acted synergistically to up-regulate CDiic in R1 cells, but no elevation occurred in R2 cells. In summary, CDlic up-regulation requires both RA and cAMP signals and is correlated to functional and morphological cell maturation. Each cell line exhibits specific signal requirements for maturation. In NB4 cells, RA and cAMP cooperate in upregulating integrins (Fig. 2) and inducing maturation (Fig. 1B), though a high concentration of RA (1 uM) is sufficient to induce maturation. R1 cells require cAMP to mature and, for full induction of CD11c, RA and cAMP acting synergistically. In R2 cells, data suggest that defective RA signaling enforced a maturation blockade by depriving cells of regulatory component(s) required for cAMP to act. (ii) NB4 cells did not require elevation of cAMP above basal to mature. However, the RA concentration required (1 ,pM) was 100- to 500-fold of the physiological plasmatic levels. Lower RA concentrations reportedly allowed the emergence of RA resistant clones. In an attempt to prove that cA-kinase not only triggered R1 cell maturation but also could contribute to maturation of sensitive cells, we took advantage of sulfur-substituted cAMP (24, 25, 27, 29). NB4 cell maturation was potentiated by low concentrations (75 ,pM) of a stable cA-kinase agonist, (Sp)-8CI-cAMP[S], even at low RA doses (Fig. 3A). This cAMP analog also triggered R1[+] cell maturation (data not shown), reproducing the effect of 8-CPT-cAMP. Synergistic effects of (Sp)-8ClcAMP[S] on NB4 cell maturation operated on a wide range of RA concentrations (Fig. 3A). Competition experiments using Sp and its antagonist diastereoisomer, (Rp)-8ClcAMP[S], were carried out (Fig. 3B). The Rp form (300 ,uM) completely abolishes the response induced by the Sp diastereoisomer (75 juM), providing evidence that maturation trig-

expression of RA-induced cellular markers but no maturation. (ii) Maturation triggering is defined by growth arrest and expression of morphological and functional features. According to this model, before a subsequent signal triggers maturation, priming by RA is required. Consistent with this proposal, resistant cells treated with RA should progress toward maturation, though no morphological change was detectable. Kinetics of induction by cAMP of mature cell functions, like NBT reduction, were evaluated in NB4, R1[+], and R1[-J cells, with and without a prior RA treatment (Fig. 1). Faster expression of the NBT-reduction function found for R1[+] cells, compared to R1[-] cells, corroborated morphological maturation, indicating that RA priming drives cells to a turning point where proliferation can be stopped and maturation can be triggered by a cAMP-responsive mechanism. Defects in RA Signaling and not cAMP Signalig Renders R2 Cells cAMP Unresponsive. Conceivably, if RA- and cAMPsignaling pathways are actively cooperating in APL cell maturation, defects located in either RA or cAMP pathways should impair cell maturation. Thus, resistant cells should be selected in the presence of both inducers. This allowed us to isolate the R2 cell line from the R1 cell line. For the purpose of comparison with NB4 and R1 cells, the features of R2 cells in response to cAMP-elevating agents are shown in Table 1. Interestingly, R2 cells were resistant to any of the cA-kinase activators tested. RA could permit cAMP-induced maturation of NB4 and R1 cells by up-regulating the cA-kinase or sensitizing it to cAMP. Conversely, in the R2 cells, the blocked cAMP effect could be due to subresponsive kinase, as we recently found in a mutant rat promyelocytic cell line selected for cAMP resistance (26). However, the level of cA-kinase (0.8 pM) and its cAMP responsiveness (Ka = 0.18 ,M) were similar in all cell types whether treated with RA (1 uM for 72 h) or not. The cA-kinase isozyme distribution (70-75% type I and 25-30% type II) was also similar. The only difference in cA-kinase expression was that RA caused a moderate induction of RI expression of both the mRNA and protein level in NB4 and not in R1 and R2 cells (data not shown). Therefore, RA pretreatment (priming) of R1 cells appeared to enable cAMPdependent step(s) distal to activation of cA-kinase; R2 cells may lack components normally issued during priming and required for cAMP to act. Evidence for Interdependence ("cross-talk") Between the RA and cAMP Signaling Pathways. Cooperation of RA and cAMP for maturation triggering was further investigated

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FIG. 1. cAMP induces maturation of RA-resistant NB4 cells. R1[+] cells were pretreated by 1 PM RA for 7 days (primed). Rl[-] cells were unpretreated controls. To test the effect of cAMP on maturation, cells were then incubated with 1 pAM RA alone or in combination with cAMP at indicated concentrations. Cell maturation was evaluated by an NBT-reduction assay (27). At least 500 cells were scored. Dose-response experiment of RAr[+] cells to cAMP (72 h of cAMP incubation) (A). Time course of NBT response of NB4, R1[+], and R1[-] cells to 100 PtM (B) and 200 pM (C) 8-CPT-cAMP. (B) R1[+] cells rapidly responded to cAMP triggering, whereas R1[-] cells did not. Response of NB4 cells treated simultaneously with the two inducers show a faster kinetic, and the R1[+] cell response was similar to the NB4 cell response to RA alone. (C) NB4 and R1[+] cells showed similar kinetics when cAMP levels were raised to 200 juM.

Cell Biology: Ruchaud et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

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with both RA (0.1 uM) and increasing doses of the selective cA-kinase antagonist (Rp)-8Cl-cAMP[S] (Fig. 3C), a significant decrease (50%) in NB4 cell maturation was observed. That maturation was partly counteracted by (Rp)-8C1cAMP[S] (competition with endogenous cAMP) suggested that the cA-kinase might be also involved in NB4 cell maturation. However, this analog failed to completely abolish maturation ofNB4 cells, even when high doses were used. It should be noted that in liver (Rp)-cAMP[S] analogs also only partially inhibit the basal cA-kinase activity (42). In conclusion, this study has shown that disrupted RA signaling completely prevents cAMP-triggered maturation. Increased cAMP is essential for maturation of R1 cells primed with RA. NB4 cells treated with very high RA will eventually mature without increased cAMP but may still depend on basal cA-kinase activity.

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8431

We have shown an absence of maturation of "resistant" APL cells at high RA molarity, though RA signaling is clearly not defective. Other myeloid leukemia cell lines resistant to RA have been described, but NB4-R1 cells differ from these in several important respects. In the non-APL HL60-R cells (31), RA signaling was abolished by a point mutation in RARa conferring dominantly negative activity. R1 cells also differ from the NB4-306 resistant cells, derived from NB4 cells after exposure to low-dose radiation (32), that fail to respond to RA (no CD11b-c up-regulation and no CD33 down-regulation). Though R1 cells and other cell lines share an absence of maturation in response to RA, the molecular mechanisms behind their failure to mature differ: these cells are unique in that they clearly respond to RA and mature provided RA and cAMP signals are combined. In contrast, the R2 clone, derived from R1 cells under the selective pressure of cAMP, shows defects in its RA response (Fig. 2 and E.D., unpublished data). Clearly, R2 cells were unresponsive to RA and, despite cA-kinase activation, maturation did not occur. These cell lines with complementary features show how crucial the interplay between RA and cAMP signaling is for maturation. Differentiation of APL is dissociated into two distinctly regulated events, priming and triggering of maturation, which

+ cAMP 48h

FIG. 2. Flow cytometry analysis of CD18/CD11c integrin expression. Numbers corresponding to the various treatments on either inducible or resistant cells are identical in A and B. [+], Resistant cells cultured with 1 ,uM RA for a week before the experiment (i.e., RA priming); [-], cells without prior RA treatment (no RA priming). Shadowed areas indicate a negative response to treatments. The various cell lines were treated with 100 ,M 8-CPT-cAMP or 1 ,M RA or simultaneously with the two inducers. When RA and cAMP were used simultaneously, cAMP was present for the last 48 h of culture. The regulation of CD18 and CD11c expression required distinct signals (RA or RA plus cAMP) depending on cell types. (A) Modulation of CD18 expression: Basal level of expression (100 arbitrary units). (B) Modulation of CD11c expression. Note the absence of up-regulation in treatments 10 and 11, and the absence of synergy of the two inducers in treatment 14, compared to the strong cooperation observed for treatment 8.

gering requires cA-kinase activation. In addition, it was noticed that an excess of (Rp)-8Cl-cAMP[S] (>300 AM) reduced the NB4 cell maturation below the level obtained with RA alone (Fig. 3B). Moreover, when cells were treated

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FIG. 3. Effects of (Sp)- and (Rp)-8CI-cAMP[S] on RA-dependent NB4 cell maturation (A). NB4 cells were treated with RA (1 nM-i ,uM), with (m) or without (o) 75 MM (Sp)-8CI-cAMP[S] for 48 h. Positivity was evaluated by counting 500-1000 cells. (B) NB4 cells were treated for 48 h with 0.1 MM RA plus 75 MuM (Sp)-8CI-cAMP[S] and increasing concentration of (Rp)-8CI-cAMP[S] to test the antagonistic effect of the Rp isomer on cA-kinase-dependent maturation. The control culture stimulated with RA alone (0.1 ,uM) showed 56% positive cells; the addition of 75 AM (Sp)-8CI-cAMP[S] increased the number of positive cells to 79%6. When cells were incubated with fixed concentrations of RA (0.1 MM) and (Sp)-8CI-cAMP[S] (75 pAM), increasing the concentration of the Rp antagonist decreased the positivity, until the agonist effect was totally blocked at 300 pM Rp. Significantly, 500 ,uM Rp-8CI-cAMP[S] decreased positivity (48%) below the positivity in the RA-stimulated control (56%). (C) (Rp)-8C1-cAMP[S] partially counteracts the RA-induced maturation response. Cells were incubated for 48 h with a constant concentration of RA (0.1 MM) and increasing concentrations of (Rp)-8CI-cAMP[S]. The inhibitory effects of (Rp)-8C1-cAMP(S] were significant from 60 uM; positivity was as low as 27% for 500 pM (Rp)-8Cl-cAMP[S], which corresponded roughly to a 50%o block of RA response.

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depend on distinct signaling pathways. cAMP is likely to control an as yet unknown pivotal event, linking priming and triggering. Uncoupled priming and triggering is a likely cause for "resistance." Cooperation of cAMP and retinoid signals reportedly plays a key role in differentiation, embryogenesis, and neoplasia (refs. 30 and 33-35; for review, see ref. 1). For instance, RA induced F9 embryo-carcinoma cells to differentiate to primitive endoderm, and then cAMP switched the differentiation pathway from visceral endoderm to parietal endoderm. By analogy, RA can be thought to operate as a priming agent in APL cells allowing a cAMP signal to induce the terminal maturation. In the APL systems, though endogenous cAMP seems sufficient for sensitive NB4 cells to mature, (Rp)-8C1-cAMP[S] studies revealed that subtle changes in cAMP levels disturbed maturation of RA-sensitive cells, suggesting that a cA-kinase activity might also be a requirement for maturation of sensitive cells. Our unexpected findings are that cAMP overrides the maturation arrest of R1 cells and that R2 cells lack the RA-dependent component(s) working in cAMP-triggered maturation. It will be important to know whether any modulation in the activity of prekinase effectors [receptor-adenyl cyclase complex (36)] or post-kinase cAMP-responsive machinery [altered protein phosphorylation or altered transcriptional response to cAMP-responsive-element binding proteins, CREBP (37) or their modulators CREM (38)] contribute to the emergence of resistant cells of the R1 type. The finding that maturation of resistant cells can be manipulated extracellularly by agonistic activation of membrane-bound hormonal receptors is promising. Finally, it remains to be determined, why maturation triggering fails in R2 resistant cells. Possible candidates are a group of proteins that fail to be induced by RA in such cells. The concept composed of a two-step RA response in NB4 cells will hopefully aid further dissection of the mechanisms underlying RA-induced maturation in APL. The NB4 sublines described in the present study should be particularly well suited for studies of the involvement in priming and triggering of RARa, the chimeric PMLRARa receptors, or other members of this family of receptors such as retinoid X receptor (for review, see refs. 2, 3, 39, and 40). S.R. and E.D. equally contributed to this work. We thank Drs. P. Chambon, R. Berger, C. J. Larsen, and D. Grausz for their help in reviewing the manuscript. This work was supported in part by Fondation contre la Leucemie and Association pour la Recherche contre le Cancer, the Norwegian Society against Cancer (Norway), and the Fonds der Chemischen Industrie and Deutsche Akademischer Austauschdienst (Germany). 1. de Luca, L. M. (1991) FASEB J. 5, 2924-2933. 2. Linney, E. (1992) Curr. Top. Dev. Biol. 27, 309-350. 3. Leid, M., Kastner, P. & Chambon, P. (1992) Trends Biochem. Sci. 17, 427-433. 4. Breitman, T. R., Selonick, S. E. & Collins, S. J. (1980) Proc. Natl. Acad. Sci. USA 77, 2936-2941. 5. Huang, M. E., Ye, C. Y., Chen, S. R., Chai, J. C., Lu, J. X., Zhoa, L., Gu, L. G. & Wang, Z. (1988) Blood 72, 567-572. 6. Chomienne, C., Ballerini, P., Balitrand, N., Daniel, M. T., Fenaux, P., Castaigne, S. & Degos, L. (1990) Blood 76, 1710-1717. 7. Rowley, J. D., Golomb, H. M. & Dougherty, C. (1977) Lancet i, 549-551. 8. Larson, R. A., Kondo, K., Vardiman, J. W., Butler, A. E., Golomb, H. M. & Rowley, J. D. (1984) Am. J. Med. 76, 827-841. 9. Borrow, J., Goddard, A. D., Sheer, D. & Solomon, E. (1990) Science 249, 1577-1580.

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