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and Retinoid-Independent Pathways
Laboratory of Molecular Biology, Centro Catullo e Daniela Borgomainerio (M.G., M.F., M.M.B., I.R., L.D., M.K., M.Te., E.G.) and Department of Oncology (M.Ta.), Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy; Sigma-Tau Industrie Farmaceutiche Riunite, Pomezia, Italy (C.P., P.C.); Dipartimento di Scienze Molecolari Agroalimentari, Università degli Studi di Milano, Milano, Italy (L.M., S.D.); and Institut de Genetique et Biologie Moleculaire, Illkirch, France (C.R.-E., E.P.)
Received February 23, 2006; accepted June 15, 2006
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Abstract |
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agonists than ATRA. We used three teratocarcinoma cell lines to evaluate the significance of RAR in the activity of RRMs: F9-wild type (WT); F9-/-, lacking the RAR gene; F951, aF9-/-derivative, complemented for the RAR deficit. Similar to ATRA, ST1926 and CD437 activate cytodifferentiation only in F9-WT cells. Unlike ATRA, ST1926 and CD437 arrest cells in the G2/M phase of the cell cycle and induce apoptosis in all F9 cell lines. Our data indicate that RAR and the classic retinoid pathway are not relevant for the antiproliferative and apoptotic activities of RRMs in vitro. Increases in cytosolic calcium are fundamental for apoptosis, in that intracellular calcium chelators abrogate the process. Comparison of the gene expression profiles associated with ST1926 and ATRA in F9-WT and F9-/-indicates that the RRM activates a conspicuous nonretinoid response in addition to the classic and RAR-dependent pathway. The pattern of genes regulated by ST1926 selectively, in a RAR-independent manner, provides novel insights into the possible molecular determinants underlying the activity of RRMs in vitro. Furthermore, it suggests that RAR-dependent responses are relevant to the activity of RRMs in vivo. Indeed, the receptor hinders the antitumor activity in vivo, in that both syngeneic and immunosuppressed SCID mice bearing F9-/- tumors have increased life spans after treatment with ST1926 and CD437 relative to their F9-WT counterparts.
; Garattini et al., 2004b). The activity of classic retinoids is the consequence of selective antiproliferative and cytodifferentiating effects that are mediated predominantly by specific nuclear receptors of the RAR and RXR families (Altucci and Gronemeyer, 2001). In many cell types, classic retinoids exert unique actions of oncological interest, including growth inhibition, cytodifferentiation, and direct or indirect cytotoxicity. Because the molecular mechanisms of action of retinoids are generally different from those of other antineoplastic agents, there is growing interest in the synthesis of molecules with new properties (Rishi et al., 2003; Sabichi et al., 2003; Cao et al., 2004; Chun et al., 2005).
A novel series of synthetic retinoic acid derivatives [retinoid-related molecules (RRMs) or atypical retinoids], with activity in leukemia and cancer cells, has been described recently (Mologni et al., 1999; Ponzanelli et al., 2000; Sun et al., 2002; Zhang et al., 2002; Garattini et al., 2004a; Lopez-Hernandez et al., 2004; Zuco et al., 2004). In certain cellular contexts, these molecules do not show cross-resistance with ATRA (Marchetti et al., 1999) and other chemotherapeutics (Ponzanelli et al., 2000), suggesting novel features and mechanisms of action relative to classic retinoids and the available anticancer agents. The prototypes of RRMs are CD437 and ST1926 (Garattini et al., 2004). CD437 is a retinoid originally developed as a selective RAR agonist (Delescluse et al., 1991). Although a preliminary report suggested that ST1926 is not an efficient RAR agonist (Cincinelli et al., 2003), we have provided evidence that the compound does bind to and trans-activate the receptor (Garattini et al., 2004b). ST1926 is a more powerful antileukemic and anticancer agent (Cincinelli et al., 2003; Garattini et al., 2004a) with better toxicologic and pharmacokinetic profiles than CD437. ST1926 is under preclinical development in view of phase I clinical trials.
Various aspects of the molecular mechanisms of action of RRMs remain unclear. From a structural standpoint, RRMs are classified as synthetic retinoids. However, the contribution of nuclear retinoic acid receptors to the antineoplastic activity of ST1926 and CD437 is not completely defined. In particular, involvement of RAR is debated (Hsu et al., 1997; Holmes et al., 2000; Sun et al., 2000a; Zhao et al., 2001). In certain cells, pharmacological inhibition of RAR blocks the action of CD437 (Holmes et al., 2000), whereas in others, this is ineffective (Mologni et al., 1999). Synthesis of an active RRM with strong apoptotic properties and no RAR binding activity (Dawson et al., 2001) adds to the controversy. Furthermore, although the activity of ST1926 and CD437 in myeloid leukemia is predominantly the result of an apoptotic process, RRMs also inhibit growth and induce cytodifferentiation in other tumor contexts. These two further actions may contribute to the overall antineoplastic activity of RRMs and have never been the object of a systematic study. Finally, the gene expression program set in motion by RRMs and its dependence on the activation of RAR or other RAR isotypes is unknown.
F9 teratocarcinoma cells represent a useful cell-autonomous model to study the activity of retinoids (Boylan et al., 1993; Taneja et al., 1997; Faria et al., 1999; Rochette-Egly et al., 2000a,b; Rochette-Egly and Chambon, 2001; Zhuang et al., 2003; Bour et al., 2005). These cells undergo growth arrest and differentiation along the primitive endoderm in response to ATRA and other synthetic retinoids. Moreover, studies conducted with F9 sublines presenting genetic deletion of RAR (Boylan et al., 1993) demonstrated that endodermal differentiation requires activation of the receptor. Thus, the F9 model is well suited to define the relative contribution of cytodifferentiation and growth inhibition to the overall antineoplastic activity of RRMs. Furthermore, the use of F9 teratocarcinoma cells is likely to provide information as to the relevance of the classic RAR/RXR and alternative or complementary pathways for the pharmacology of RRMs. Finally, the availability of F9 teratocarcinoma cell lines differing only in the expression of RAR (Taneja et al., 1997) represents a unique tool to study the significance of the receptor for the antineoplastic activity of RRMs. The growth of F9 teratocarcinoma cells as solid tumors in syngenic strains of animals is a further advantage, permitting in vivo experiments.
In this study, three F9 teratocarcinoma cell lines with different expression of RAR were used to investigate the cytodifferentiating, antiproliferative, and apoptotic effects of ST1926 and CD437 compared with ATRA. In the same system, using whole-genome microarrays, ST1926 specific responses and their RAR-dependence were defined. Finally, the involvement of RAR in the overall antitumor activity of RRMs was studied in normal and immunosuppressed mice transplanted with F9 cells.
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Materials and Methods |
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Cell Cultures and Transfections. COS-7 (American Type Culture Collection, Manassas, VA) and the F9 teratocarcinoma cell lines, F9-WT, F9-/-, and F951 (Taneja et al., 1997), were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and were free from mycoplasma. COS-7 cells were transfected with human RAR
, RAR
, RAR, or RXR pSG5-based plasmids along with the DR5-tk-CAT (RAR-dependent) or DR1-tk-CAT (RXR-dependent) reporter genes and the normalization plasmid pCH110 (-galactosidase) (Garattini et al., 2004a). Transactivation assays on the various isoforms of RAR were performed using extracts of transfected COS-7 treated for 24 h with different concentrations of the test retinoid, as described previously (Garattini et al., 2004a)
Cellular Proliferation, Viability, and Apoptosis. Cell number and viability were determined after staining with erythrosin (Sigma). For the determination of the apoptotic index, adherent cells were detached, fixed in methanol, and stained with 4,6 diamidino-2-phenylindole (Giannì et al., 2000). The apoptotic index is the percentage of cells with features of nuclear fragmentation after counting a minimum of 300 nuclei/field under the fluorescence microscope. In some experiments, apoptosis was determined according to the Annexin-V assay by flow cytometry (Mebcyto Apoptosis Kit; MBL International, Woburn, MA) (Garattinì et al., 2004a), using the flow cytometer FACSCalibur (BD Biosciences, Palo Alto, CA). Caspase-3 activation was measured with the fluorogenic peptide substrate DEVD-amc (N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin; Alexis, Laufelingen, Switzerland) (Mologni et al., 1999).
Flow Cytometric Cell Cycle Analysis. F9-WT and F9-/- cells were counted using a Coulter counter (Beckman Coulter, Fullerton, CA) and fixed in 70% ethanol. Cells (1-2 x 106) were washed with PBS and stained with 1 ml of a solution containing 10 µg/ml propidium iodide (PI) and 10,000 units of RNase overnight at 4°C in the dark. Flow cytometric analyses were performed using FACSCalibur, and the distribution of the cells in the different cell cycle phases calculated by the gaussian method (Ubezio, 1985).
RNA Preparation and RT-PCR. Total RNA was extracted according to the guanidinium-thiocyanate-cesium chloride method, reverse transcribed, and amplified by polymerase chain reaction (PCR) using synthetic amplimers (GeneAmp RNA-PCR core kit; Applied Biosystems Inc., Branchburg, NJ). The amplimers used are shown in Table 1.
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Real-time RT-PCR was performed using Taqman gene expression assays (Applied Biosystems, Foster City, CA) following the manufacturer's instructions, on a GeneAmp 5700 sequence detector (Applied Biosystems). The assays used were Mm00439359_m1, with primers located at the 1-2 exon boundary of the HOXA1 gene; Mm00487803_m1, with primers located at the 1-2 exon boundary of the c-myc gene; Mm00445212_m1, located at the 7-8 exon boundary of the KIT oncogene; Mm01319677_m1, located at the 4-5 exon boundary of the RAR gene; and Mm00435270, located at the 19-20 exon boundary of the Notch homolog 3 gene. The -actin housekeeping gene (assay ID Mm00607939_s1) was used for the normalization of the results.
Measurement of Intracellular Calcium. Changes in intracellular calcium concentrations were measured at the single-cell level in a semiquantitative fashion. In brief, F9-WT or F9-/- cells were seeded (300,000/ml) on microscopic glass slides and allowed to adhere overnight. Cells were labeled with 4 µM Fluo3-AM (Invitrogen) at 37°C for 1 h. Slides were washed twice with PBS and incubated in PBS containing 1.26 mM CaCl2. After addition of vehicle (DMSO) or ST1926 (1 µM) the associated fluorescence was measured for a maximum of 1 h with an IX70 microscope (Olympus, Hamburg, Germany) equipped with an imaging system (Till Photonics GMBH, Gräfelfing, Germany). For each experiment, cells were scanned for at least 30 s to establish a baseline fluorescence reading before addition of the appropriate stimulus. All incubations were carried out while continuously scanning the cells every 200 ms.
For the quantitative determination of intracellular calcium in the entire cell population, a previously described protocol was used (Garattini et al., 2004a). For these experiments, the FURA-2-associated fluorescence was measured continuously at 37 ± 1°C with a spectro-photofluorometer (LS-50B; PerkinElmer Life and Analytical Sciences, Milano, Italy).
In Vivo Studies. F9-WT or F9-/- cells (3 x 106) were inoculated intraperitoneally in 129/Sv syngenic animals or in immunodeficient SCID mice (Charles River Italia, Calco, Italy). ST1926, CD437, or ATRA was dissolved in a diluent consisting of cremophor/ethanol (Garattini et al., 2004a). All the compounds were administered i.p. or orally once per day for up to 3 weeks. Body weight and lethality were recorded every day. All the experiments were approved by the Internal Animal Care Committee and conducted according to the pertinent International and Italian legislation.
Gene Microarrays. F9-WT and F9-/- were treated for 10 h with vehicle (DMSO), all-trans-retinoic acid (0.5 µM), or the synthetic retinoid ST1926 (0.5 µM). The polyadenylated RNA fraction was isolated from total RNA using magnetic oligo(dT) micro-spheres (DYNAL AS, Oslo, Norway). Polyadenylated RNA was amplified and labeled with the fluorochrome Cy3 or Cy5 using the Amino allyl MessageAMP II kit (Ambion Inc., Austin, TX). Each RNA preparation represents a pool of four flasks treated independently with the appropriate stimulus. Each experimental sample consists of 1) a replicate in which the control RNA is labeled with Cy3 and the treated RNA with Cy5 and 2) a swapped replicate in which the control RNA is labeled with Cy3 and the treated RNA with Cy5. An equal amount of Cy3- and Cy5-labeled cRNAs were mixed and hybridized to the oligonucleotide microarray (Agilent 60-mer microarray oligo processing protocol version 2.1; Agilent Technologies, Palo Alto, CA). After washing, microarray glass slides were scanned with the G2565AA dual laser scanner (Agilent Technologies). Images were analyzed with the Feature Extraction software version 7.1 (Agilent Technologies). Dye normalization was performed automatically, using the Rank Consistency filter and the LinearLOWESS normalization method. The raw data of the microarray experiments were deposited in the public database Miamexpress (http://www.ebi.ac.uk/miamexpress) with the accession number E-MEXP-361. Further Qspline normalization (Workman et al., 2002) of the intensity data was conducted using Gene Publisher (Knudsen et al., 2003). Two-way ANOVA based on F distribution (factor A: vehicle, ST1926, or ATRA; factor B: F9-WT or F9-/-) was performed with the T-MEV software (Saeed et al., 2003; http://www.tm4.org/mev.html). Significant genes (p < 0.0001) were classified based on Pavlidis template-matching algorithm and hierarchical clustering using Pearson correlation as the distance measure (Pavlidis and Noble, 2001; Kasturi et al., 2003).
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Results |
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Activators. We compared the ability of ST1926, CD437, and ATRA to activate RAR, RAR, and RAR overexpressed in COS-7 cells (Table 2). The EC50 reflects the affinity of each retinoid for the three isoforms of RAR. For RAR and RAR, the rank order of potency was ATRA = CD437 > ST1926, whereas for RAR, it was CD437 > ATRA > ST1926. RAR selectivity was calculated as the inverse ratio of the EC50 values. The data obtained are consistent with previous results, indicating that ATRA was a nonselective (pan-RAR) agonist, whereas CD437 was a selective RAR agonist. In addition, our results demonstrated that ST1926 bound and trans-activated the three receptors with similar affinity and, unlike CD437, lacked RAR selectivity. Table 2 also shows the maximal activity of each compound at saturating concentrations. It is noteworthy that both ST1926 and CD437 activated RAR more efficiently than ATRA. Cotransfection of RXR did not change the EC50 values of ST1926, CD437, and ATRA for the various isoforms of RAR (data not shown). This is in line with the concept that COS-7 cells contain significant amounts of native RXRs that combine with overexpressed RARs to form transcriptionally active RXR/RAR heterodimers (Garattini et al., 2004b; Giannì et al., 2006).
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ST1926 and CD437 are unlikely to possess significant RXR agonistic activity, as demonstrated by transfection of COS-7 cells with RXR and an appropriate reporter gene activated by RXR/RXR homodimers. Under these experimental conditions, the RXR agonist 9-cis retinoic acid (1 µM) caused a 9.7 ± 0.8 (mean ± S.D., n = 3) induction of CAT reporter activity over the unliganded control sample. In contrast, the effects of ST1926 or CD437 were minimal, dose-independent, and did not exceed a 2-fold induction at all the concentrations investigated (0.01 µM-10 µM).
The F9 Teratocarcinoma Model. To define the relevance of nuclear retinoic acid receptors, and RAR in particular, for the pharmacological activity of ST1926 and CD437, we chose the mouse F9 teratocarcinoma model. Experiments were performed on three clonal cell lines with different expression of RAR. As shown in Fig. 1A, wild-type F9 cells (F9-WT) expressed RAR and RAR constitutively. As expected, RAR was not expressed under basal conditions. Ablation of the RAR gene by homologous recombination results in an F9 subline (F9-/-), which maintains synthesis of RAR but has an absolute deficit of RAR (Boylan et al., 1993). RAR expression in the F9-/- cellular background is partially reconstituted by transgenesis in F951 cells (Taneja et al., 1997).
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-/- cell lines in standard culture conditions were very similar. Furthermore, the growth of the two cell types did not differ significantly from that determined for RAR51. Finally, the morphology of the three lines growing in basal conditions is indistinguishable (data not shown). The similarities of F9-WT and F9-/- cells in vitro are recapitulated in vivo. Figure 1C illustrates the growth characteristics and the morphology of the tumors derived from the F9-WT and F9-/- cells in syngeneic 129/Sv mice. The two types of tumors grew as homogeneous solid masses in the peritoneal cavity and had similar and highly undifferentiated cell morphology. The growth kinetics of the two tumors were superimposable and resulted in masses of similar weight and volume. The median survival times of animals inoculated with F9-WT and F9-/- cells did not differ significantly and were 14.3 ± 3.2 and 13.0 ± 3.1 days (mean ± SD of three independent experiments), respectively. All together, our results indicate that RAR expression had no influence on the in vitro or in vivo growth and morphology of untreated F9 cells. These features support the relevance of the model for our comparative studies on RRMs and ATRA.
ST1926 and CD437, Like ATRA, Induce F9 Cytodifferentiation via RAR. To study cytodifferentiation, we compared the ability of ATRA, ST1926, and CD437 to induce the expression of the mRNAs coding for two validated markers of the primitive endoderm, collagen IV and laminin B in F9-WT and F9-/- (Fig. 2A). As expected, the synthesis of the collagen IV and laminin B transcripts was induced in F9-WT cells treated with ATRA (0.1 µM) for 48 h but not in the F9-/- counterpart. Both ST1926 and CD437 (0.1 µM) were at least as effective as ATRA in inducing the two marker mRNAs; once again, this phenomenon was evident only in the F9-WT cell line. Hence, under the experimental conditions considered, the two RRMs acted as classic retinoids and induced primitive endodermal differentiation in a RAR-dependent mode. Our results are consistent with the fact that RAR expression is necessary for the retinoid-dependent differentiation of F9 into primitive endodermal cells (Boylan et al., 1993; Plassat et al., 2000). More importantly, they demonstrate that ST1926 and CD437 activated RAR in a natural cellular context and behaved like classic retinoids at the concentration considered.
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ST1926 and CD437 Induce Growth Arrest and Cell Death via a RAR-Independent Mechanism. We evaluated whether the RAR-dependent cytodifferentiation induced by the two RRMs was accompanied by growth inhibition. As documented by Fig. 2B, the action of ST1926 and CD437 could be divided in two phases. In fact, within the first 48 h, both compounds blocked the growth of F9-WT and F9-/- cells. At first, growth inhibition was not accompanied by a loss in cell viability, which was always above 80%, as observed in the case of control cultures (data not shown). After 2 days of treatment, a progressive and similar decrease in viability was evident in both cell lines. Contrary to what was observed in the case of the two RRMs, equimolar concentrations of ATRA induced only a delay in the growth of F9-WT and F9-/- cells and no significant cytotoxicity at any of the time points considered. As shown in Fig. 2C, increasing the concentration of ST1926 and CD437 to 2 µM caused a dose-dependent decrease in the number of viable cells already evident at 48 h. In these conditions, ST1926 and CD437 reduced the viability of F9-WT cells from 88 ± 1% (mean ± S.D., n = 3) to 66 ± 2 and 63 ± 6 at 0.5 µM, 29 ± 4% and 41 ± 10% at 1 µM, as well as 12 ± 4% and 6 ± 2% at 2 µM. Even at these relatively high concentrations, the cytotoxic effect of the two compounds did not correlate with RAR expression. In fact, not only F9-WT and F9-/- cells but also F951 cells showed very similar responses to the cytotoxic action of ST1926 and CD437. It is noteworthy that even the highest concentrations of ATRA (1 and 2 µM), though producing a growth inhibitory effect (Fig. 2C), continued to be devoid of significant cytotoxic activity on any of the cell lines considered. In fact in all cases, cell viability did not differ significantly from control conditions (data not shown). Overall, our results indicate that, in the case of both the RRMs and ATRA, growth inhibition was dissociated from the induced cytodifferentiating action. More importantly, they demonstrate that the observed growth inhibitory effects of ST1926 or CD437 and ATRA were intrinsically different. ATRA showed a pure antiproliferative action, whereas the activity of the other two compounds on F9 cell growth involved both an antiproliferative and a cytotoxic component.
ST1926 and CD437 Induce a G2/M Arrest of the Cell Cycle. The cell cycle perturbations afforded by low concentrations (0.1 µM) of the two RRMs and ATRA were determined within the first 24 h of treatment. These experimental conditions were designed to avoid interferences caused by cytotoxicity. Figure 3 shows FACS profiles, along with the corresponding quantitative results, of F9-WT and F9-/- cells treated for 12 and 24 h with the three compounds. The overall picture of the cell cycle perturbations afforded by ST1926 and CD437 was similar in both F9 lines. The two compounds caused a rapid depletion of the G1 compartment that was already evident at 12 h. This was accompanied by an expansion of the S and/or G2/M phase. The results obtained with ATRA were significantly different. Despite a slowed progression along the cell cycle, indicated by an increase in the S phase at 24 h, ATRA caused no accumulation in G2/M in either F9-WT or F9-/- cells. The expected accumulation in G1, as demonstrated in previous reports (Li et al., 2004), was observed at later time points (3-4 days, data not shown). It was clear that ST1926/CD437 and ATRA blocked the cells in two distinct phases of the cycle, demonstrating that the cellular mechanisms activated were different. Thus, our results support the concept that a classic retinoid dependent response was not at the basis of the early growth arrest afforded by RRMs.
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ST1926 and CD437 Were Characterized by an Apoptotic Action That Did Not Require RAR. As shown in Fig. 4A, treatment of F9-WT, F9-/-, and F951 cells with ST1926 for 24 h induced morphological changes characteristic of apoptosis in a dose-dependent manner and regardless of RAR expression. This was accompanied by the appearance of early apoptotic markers, such as annexin V binding to the plasma membrane (Fig. 4B) and caspase-3 activation (Fig. 4C). The fraction of viable (annexin V and PI negative, AV-/PI-), early apoptotic (AV+/PI-), late apoptotic (AV+/PI+), and necrotic (AV-/PI+) cells present in F9-WT and F9-/- cultures was evaluated by flow cytometry, after treatment with ST1926 or ATRA (0.5 µM) for 24 h. Fig. 4B, left, shows typical FACS scatter plots obtained upon challenge of F9 cells with vehicle or ST1926. A summary of the quantitative data is presented in bar graphs. In both F9-WT and F9-/- cultures, ST1926 caused a similar increase in the proportion of early and late apoptotic cells, leaving the number of necrotic cells basically unaltered. This was mirrored by a proportional decrease in the number of viable cells in ST1926 treated F9-WT and F9-/- cultures. Exposure of F9-/- and F9-WT cells to 0.5 µM ST1926 or CD437 for 24 h resulted in a similar activation of the early apoptotic marker, caspase-3, as measured by hydrolysis of the DEVD-amc substrate (Fig. 4C). Taken together, our results indicate that apoptosis was the main modality of the observed RRM-induced cytotoxic effect.
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-/- cell cultures exposed to 1 µM ATRA for 6 days contained 90 ± 1% live, 4 ± 1% early apoptotic, 1 ± 0.2% late apoptotic, and 4 ± 1% necrotic cells, compared with 92 ± 1% live, 5 ± 1% early apoptotic, 1 ± 0.1% late apoptotic and 3 ± 1% necrotic cells in the corresponding vehicle-treated cell cultures. Similar results were obtained in F9-WT cells (data not shown). Thus, in conditions of complete cell-kill by ST1926 and CD437, ATRA did not show apoptotic or cytotoxic activity. The data indicate that RAR expression neither mediated nor modulated RRM-induced programmed cell death. In addition, they suggest that activation of the retinoic acid nuclear receptor pathway is unlikely to be significant for the apoptotic process activated by RRMs.
The Apoptotic Response of F9 Cells to RRMs Involved an Early Rise in Cytosolic Calcium. One of the earliest effects activated by RRMs in the NB4 leukemia cell line was is an increase in cytosolic calcium ions (Garattini et al., 2004a). We proposed that the phenomenon is at the basis of the apoptotic process activated in this cell line. To establish whether perturbations in the homeostasis of calcium are a general phenomenon, we performed a number of experiments in the F9 model.
In a first set of experiments (Fig. 5A), the amounts of intracellular calcium were determined semiquantitatively in single F9-WT cells grown as monolayers and preloaded with the calcium indicator Fluo3-AM. Treatment of F9-WT cells for up to one h with ST1926, but not vehicle (data not shown), resulted in a significant and time-dependent increase of intracellular calcium. This effect is evident in the majority of ST1926 treated cells. Similar results were obtained in F9-/- cells (data not shown). To get quantitative information on the phenomenon, FURA-2 was measured continuously on suspensions of F9-WT and F9-/- cells after addition of ST1926 or CD437 (Fig. 5B). Treatment with the two RRMs leads to an immediate increase in the cytosolic levels of calcium with very similar time courses. The time dependence values for the elevation of FURA-2 fluorescence induced by ST1926 and CD437 in the two cell lines are very similar. At equimolar concentrations, ATRA (Fig. 5B, right) and inactive ST1926 congeners (data not shown) exerted no significant action on calcium homeostasis. Our data demonstrate that RRMs induced a rapid and long-lasting elevation of cytosolic calcium that is independent of the adherence to an extracellular substratum. Furthermore, they suggest that calcium mobilization represents an upstream event in the biochemical cascade activated by ST1926 and CD437 in F9 cells. In particular, RRM-dependent increases in cytosolic calcium preceded the appearance of any sign of apoptosis. As documented by Fig. 5C, the intracellular calcium chelator BAPTA (10 and 50 µM) suppressed the elevation of calcium afforded by ST1926 or CD437 in both cell lines. Figure 5D demonstrates that BAPTA (10 µM) blocked the RRM-dependent caspase-3 activation observed in F9-/- and F9-WT cells. These results indicate a central role for calcium, even in the case of a transcription and protein synthesis-dependent process of apoptosis like the one activated by RRMs in F9 cells.
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-/- Cells Indicates Activation of Retinoid-Dependent and -Independent Pathways by RRMs. F9-WT and F9-/- cells were used to compare perturbations of the transcriptome induced by equimolar concentrations of ST1926 and ATRA. With this type of experiment, we aimed at gathering information on the molecular mechanisms underlying the action of ST1926. In particular, we intended to identify the gene profiles associated with the retinoid-dependent and -independent components of the RRM action. Furthermore, we wanted to establish the fraction of genes whose expression is controlled or modulated by RAR activation.
As shown in Fig. 6A, treatment of F9-WT and F9-/- cells with ST1926 or ATRA (0.5 µM) for 10 h results in a significant up- or down-regulation of 2523 probes (2296 genes) (p < 0.0001 after two-way ANOVA for any of the two factors, cell line or treatment). A total of 903 probes (847 genes) has regulation patterns relevant to the study and can be classified in nine groups after Pavlidis template matching (Pavlidis and Noble, 2001) and hierarchical clustering (Fig. 6 and Fig. 7). A complete list of these genes and their functional classification (using Ease; http://david.niaid.nih.gov/david/ease1.html) is presented in Supplementary Table 2. The diagram illustrates the number of genes falling in the various classes defined. Of the 178 probes under the control of ATRA (Fig. 6B, groups 1-6), 101 (57%) were regulated in a similar manner by ST1926 (groups 1-3). This supports the concept that the RRM is a bona fide retinoid and activates a substantial fraction of the same genetic program controlled by ATRA. However, our results also demonstrated that ST1926 regulated a much larger set of genes than ATRA in both F9-WT and F9-/- cells (826 versus 178; Fig. 6A). This indicates a major contribution of retinoid-independent pathways to the early action exerted by ST1926 on the F9 transcriptome.
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Genes Regulated Concomitantly by ST1926 and ATRA Defined Classic Retinoid Responses. Group 1 includes target genes up- or down-regulated concomitantly by ST1926 and ATRA in both F9-WT and F9-/- cells (Fig. 6B). This group consisted of genes possibly modulated by ligand-induced activation of RAR/RAR. Among the genes whose expression was diminished, Myc and the DNA methyltransferase 3B stand out. Myc is a gene down-regulated by retinoids in different cellular contexts and is known to control progression from the G1 to the S phase of the cycle. Myc down-regulation may play a role in the cell cycle arrest triggered by ATRA in G1 but is unlikely to be a major determinant of the G2/M arrest observed in the case of ST1926. DNA methyl transferases are associated with gene silencing, and their inhibition has been implicated in the gene activation effects triggered by classic retinoids (Fazi et al., 2005).
The cluster of genes up- or down-regulated by ST1926 and ATRA in F9-WT cells preferentially (Fig. 6B, group 2) was likely to be under the control of ligand-activated RAR. Homeobox A5 and iroquois-related-homeobox 2 belong to this group, suggesting a particular significance for the process of primitive endodermal maturation induced by ST1926 and ATRA. Indeed, homeotic genes are well known retinoic acid targets and are implicated in morphogenesis and organogenesis (Deschamps and van Nes, 2005). Several other members of this family were noticeably induced preferentially by ATRA in a RAR-dependent fashion (Fig. 6B, group 5). Homeobox gene A1 (HOXA1) was the only HOX gene induced specifically by ATRA not only in F9-WT but also in F9-/- cells (Fig. 6B, group 4). However, the basal levels of HOXA1 expression were much higher in the former than in the latter cell line (Miamexpress; accession number E-MEXP-361), suggesting RAR independence in the case of induced expression and possible RAR dependence in the case of basal expression.
The molecular signature associated with ST1926 and ATRA in F9-/- cells (Fig. 6B, group 3) was of unknown significance and consisted of 11 genes. The sole gene upregulated in this group was the G protein 3, a polypeptide involved in G-protein signaling.
Genes Specifically Regulated by ST1926 Defined Nonretinoid-Associated Responses. The genes modulated by ST1926, and not by ATRA, in both F9-WT and F9-/- cells (Fig. 7, group 7) are likely to be relevant for the selective in vitro apoptotic action and G2/M arrest triggered by the RRM. Consistent with this notion, 23 of the 355 up-regulated genes are proapoptotic or involved in the arrest of the cell cycle. For the same reason, 11 of the 163 down-regulated genes are positive modulators of cell growth. The list of proapoptotic genes includes elements of both the intrinsic and extrinsic pathways of programmed cell death, such as p73, caspase-3, members of the TNF receptor family, and TRAF4. As for TNF receptors, it is important to underscore that these proteins belong to the same family of the death receptors that plays a fundamental role in the extrinsic pathway of apoptosis. CD437 has been proposed to exert its apoptotic action through activation of autocrine or paracrine loops involving death receptors and corresponding ligands, such as the FAS and FAS-ligand couple (Sun et al., 2000b; Jin et al., 2005). In group 7, PERP, APAF1, and Bcl-2 binding component 3, are direct or indirect targets of p53. It is noteworthy that F9 teratocarcinoma cells are known to express wild-type p53 (Mayo and Berberich, 1996), which sensitizes other types of neoplastic cells to the action of RRMs (Sun et al., 1999). Given the importance of calcium in the process of apoptosis triggered by RRMs, the presence of 19 genes encoding polypeptides, such as protein phosphatase 1D and diacylglycerol kinase , regulated by the cation, was of particular significance. The modulation of these genes might be the consequence of the early increase in calcium ions afforded by ST1926 and CD437. Group 7 contained a number of induced genes involved in DNA repair (four genes, six probes; Supplemental Table 1), suggesting that ST1926 may have DNA damaging activity (Garattini et al., 2004b).
Groups 8 and 9 consisted of genes modulated by ST1926, but not ATRA, in either F9-WT or F9-/- cells specifically. In F9-/- cells, treatment with ST1926 caused down-regulation of four genes involved in motility and angiogenesis and up-regulation of three genes responsible for cellular adhesion (Fig. 7, group 9). Conversely, the RRM induced the expression of two cell motility and angiogenesis genes (septin 2 and neuropilin) in F9-WT cells. Induction of group 8 gene products acting at the interface between the cell and the extracellular space, such as receptors of the platelet-derived growth factor or TNF family and BMP membrane bound inhibitor (Supplemental Table 1), are also of particular interest. These observations indicate that ST1926 has RAR-dependent effects on cellular processes other than proliferation, differentiation, and apoptosis.
ST1926 and CD437 Modulated the Same Type of Genes. The results obtained with whole-genome microarrays were validated and extended to CD437 for a selected number of genes. A first validation was conducted by semiquantitative agarose gel electrophoresis of the cDNA bands obtained by RT-PCR (Fig. 8A, left). For c-kit, c-myc, Notch Hom.3, and the homeo box A1 (HOXA1) gene, we also obtained quantitative results by Taqman real-time PCR (Fig. 8A, right). The experiments presented were totally independent of each other and of the ones used for the microarray analysis. The expression profile of all the genes considered was entirely consistent with what was observed with the microarray experiments. A small exception to this rule (1 of 10 genes, including RAR; see below) is represented by c-kit, which shows induction by ST1926 (and CD437) also in F9-/- cells. This may be because the primers used for the RT-PCR and Taqman real-time PCR analyses are targeted against regions of the transcripts that are different from those represented by the corresponding microarray probes. Consistent with the primary microarray data, the Taqman data demonstrate that the constitutive levels of the HOXA1 transcript are lower in the F9-/- than in F9-WT cells. It is noteworthy that all these genes were regulated by CD437 in a fashion similar to that of ST1926, suggesting that the two compounds activate a very similar genetic program. This further supports the concept that ST1926 and CD437 belong to the same functional family of compounds (Garattini et al., 2004a).
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belonged to the group of genes induced by ATRA more than ST1926 in both F9-WT and F9-/- cells (group 4). As a whole, the RT-PCR data (Fig. 8B) confirmed that the receptor was induced more efficiently by ATRA than by RRMs in F9-/- cells. However, this differential effect was not observed in F9-WT cells, also because of the lower basal levels of the transcript in this cell line. Although quantitative differences in the induction of RAR by ATRA and ST1926 or CD437 were observed in F9-WT and F9-/- cells, the finding is in agreement with previous results indicating that RAR2 can be up-regulated by multiple RAR isoforms in a redundant fashion (Rochette-Egly and Chambon, 2001). It is noteworthy that RAR was the only retinoid receptor whose expression was modulated by RRMs or ATRA in F9 cells. In fact, ST1926, CD437, and ATRA did not affect the basal level of expression of RAR, RAR, or the three RXR isoforms (Fig. 8B, left).
In Vivo, the Antitumor Activity of RRMs Was Hindered by RAR Expression in the Neoplastic Cell. The microarray results provide information on the possible molecular determinants of the RAR-independent processes (growth inhibition and apoptosis) triggered by RRMs in F9 cell cultures. However, they also point to a set of RAR-dependent alterations in genes controlling processes of potential significance for the antitumor activity of RRMs in vivo, such as cell adhesion, motility, angiogenesis, proteolysis, and tumor/host interactions. Indeed, the microarray results suggest that RRM antitumor activity might be higher in the absence of RAR.
The above hypothesis was tested directly in vivo, taking advantage of the fact that F9-WT and F9-/- grow as tumors in syngeneic mice (Fig. 1C). Figure 9A demonstrates that long-term intraperitoneal administration of ATRA at the maximal tolerated dose of 15 mg/kg did not alter the survival of either F9-WT or F9-/- tumor-bearing animals. The finding indicates that the ATRA-dependent antiproliferative and cytodifferentiation effects observed in cultures of F9 cells were not sufficient to translate into a therapeutic effect in vivo. In contrast, Fig. 9B demonstrates that oral administration of ST1926 (30 mg/kg) resulted in a significant increase in the median survival time of mice inoculated with both F9-WT and F9-/- cells. Consistent with our hypothesis, ST1926 was much more effective in F9-/- than in F9-WT tumor-bearing animals. Differential sensitivity of F9-/- and F9-WT tumors was independent of the administration route, in that a similar phenomenon was observed after i.p. treatment with ST1926 (Fig. 9C). Furthermore, the phenomenon was observed also in the case of the other RRM, CD437. Immunological responses do not seem to be at the basis of the observed differences in sensitivity between the F9-/- and F9-WT tumors. This is supported by the results obtained in the B- and T-cell-deficient SCID mouse (Fig. 9D). Even in this model, ST1926 and CD437 (i.p. administered) demonstrate a superior effect on mice bearing F9-/- tumors. Thus, our results demonstrate that RAR expression hinders the response of F9 cells to the therapeutic activity of RRMs in vivo. More importantly, they suggest that the in vivo action of RRMs is not simply the result of a direct cytotoxic and growth inhibitory action on tumor cells and may involve effects on the processes of metastatization, angiogenesis, or interactions with the host environment.
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, RAR, and RAR in COS-7 transfected cells. The data indicate that CD437 was a selective ligand of RAR and a stronger agonist of the receptor than ATRA. ST1926 itself was a better RAR transactivator than ATRA but lost RAR selectivity. Both ST1926 and CD437 were poor RXR activators.
In F9 teratocarcinoma cells, we confirmed, in a native context, that ST1926 and CD437 were bona fide retinoids and had the potential to activate RAR. As expected from classic retinoids with RAR agonistic activity, low concentrations of the two RRMs were as effective as ATRA in inducing the cytodifferentiation of F9-WT but not F9-/- cells. The results obtained with whole-genome microarrays support the concept that ST1926 is endowed with classic retinoid activity. ST1926 modulates the expression of a large proportion of the genes controlled by ATRA in F9-WT and F9-/- cells. A similar proportion of coregulated genes is observed in the responses specific for the F9-WT cellular context, where ATRA and RRM dependence is likely to be mediated by RAR. These genes are of particular interest for the differentiation of teratocarcinoma cells along the parietal endoderm pathway.
The data obtained in the F9 model indicate that retinoid activity was unlikely to play a significant role in RRM-induced apoptosis, which is a primary determinant of ST1926 and CD437 antitumor activity. Indeed, the apoptotic action of RRMs is not shared by ATRA, strongly suggesting that programmed cell death is not a direct consequence of the retinoid-dependent cytodifferentiating effects as in NB4 leukemic cells (Giannì et al., 2000). Furthermore, unlike cytodifferentiation, apoptosis was independent of RAR activation, as observed in F9-WT, F9-/-, and F951 cells. Our results are consistent with the idea that ST1926 and CD437 activated not only the classic retinoid pathway but also a second signaling pathway. In cultures of F9 cells, the second pathway is prevalent and is at the basis of the apoptotic effect.
Similar to what we observed in NB4 myeloid leukemia cells (Garattini et al., 2004a), the earliest event associated with RRM-induced apoptosis in F9 cells is the elevation of cytosolic calcium. In NB4 cells, we demonstrated previously that calcium increases are not due to a net influx of the cation from the extracellular compartment and might be the result of RRM effects on the reuptake of the ion by the mitochondrion. Whatever the underlying mechanism, the rise in calcium is necessary for the programmed cell death activated by RRMs in NB4 and in F9 cells. In fact, in this last cell line, whereas ST1926 and CD437 were powerful calcium-mobilizing agents, ATRA and inactive RRMs were devoid of this activity. More importantly, chelation of intracellular calcium by BAPTA prevented ST1926-induced caspase activation, one of the hallmarks of apoptosis. Calcium mobilization was not influenced by RAR, in that identical effects were observed in the F9-WT and F9-/- cell lines. Although the mobilization of calcium observed in RRM-treated F9 teratocarcinoma and NB4 cells is similar, the ensuing process of apoptosis is intrinsically different. In NB4 cells, apoptosis is very rapid and does not require gene expression or de novo protein synthesis. In contrast, the apoptotic response of F9 cells was slower and might therefore be dependent on gene transcription and protein synthesis. If this was indeed the case, genes relevant for the RRM-dependent apoptosis in F9 cells must be sought for among the large number of probes identified as specific and RAR-independent targets of ST1926 (the genes controlled by ST1926, but not ATRA, in both F9-WT and F9-/- cells). This set of genes is of particular significance and deserves evaluation as to calcium-dependent transcriptional modulation.
The overall antitumor effect of RRMs may be the result not only of apoptotic but also of antiproliferative effects. To establish this point, we conducted studies with low and comparable concentrations of ST1926 or CD437 and ATRA. Under these conditions, the process of apoptosis activated by the two RRMs was delayed and the antiproliferative effects could be studied in a relatively clean situation. Our data demonstrate that ST1926 and CD437 treatments were associated with an early S and G2M cell cycle block in both F9-WT and F9-/- cells. The cell cycle block afforded by ATRA is different and consists of a G1 arrest observed only after long exposures (3-4 days) (Li et al., 2004). The G1 arrest caused by ATRA in both F9-WT and F9-/- cells suggests involvement of RAR and or RAR. The assumption is in line with the observation that RAR activation is a critical determinant of the growth arrest induced by ATRA in F9 cells (Faria et al., 1999; Zhuang et al., 2003).
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is not a major determinant of RRMs' apoptotic and cytotoxic activity in vitro, the receptor seems to play an important role in the response of F9 tumor-bearing animals. Syngeneic and immunodeficient mice transplanted with F9-/- cells are more sensitive to RRMs than the corresponding counterparts inoculated with F9-WT cells. The phenomenon is not explained by differences in basal growth, in that the F9-WT and F9-/- untreated tumors grow in a similar fashion, show the same histological appearance, and are lethal to animals in approximately the same amount of time. Furthermore, the phenomenon is unlikely to reflect clonal variability between the F9-WT and F9-/- cells. In fact, animals transplanted with the two cell lines are equally insensitive to ATRA, whereas they respond equally well to intraperitoneal injection of other chemotherapeutic agents, such as cisplatin (M. M. Barzago and E. Garattini, unpublished observations). Although originally unexpected, this increased sensitivity may find an explanation in the profile of genes selectively modulated by ST1926 in either F9-WT or F9-/-. In fact, the cell type-specific effects observed on a number of genes controlling cell motility and angiogenic responses suggest relevance for the processes of invasion and metastasis. Overall, our data indicate that RAR expression modulates some aspects of the host/tumor interaction unrelated to immune responses.
Figure 10 provides a summary and a rational interpretation of our observations. RRMs activated two distinct pathways in F9 cells. We defined as nonretinoid the actions exerted by ST1926 (and CD437) that were not shared by ATRA and that were likely to involve mechanisms unrelated to retinoic receptor activation. The nonretinoid pathway leads to G2/M cell cycle arrest and apoptosis, which is mediated by a perturbation of calcium homeostasis. These two processes are likely to mediate a large proportion of the in vivo antitumor activity of both ST1926 and CD437. Although RAR and RAR contribute to the genomic effects induced by ST1926 in F9 cells, activation of the two receptors does not seem to play a significant role in the overall antitumor activity of ST1926 and CD437 as well. Activation of RAR by RRMs leads to cytodifferentiation; however, this phenomenon is unlikely to have any significance for the in vivo antineoplastic action of these compounds. In contrast, RAR seems to have an important modulatory and negative role on the response to RRMs in vivo, perhaps by acting on genes involved in cell motility and adhesion.
In conclusion, the data presented provide the foundation for future studies aimed to define the functional relevance of the numerous genes modulated by RRMs. Our results also have far-reaching implications at the clinical level, in that they may orient the choice of target tumors. Indeed, if RAR-related resistance is not limited to the F9 model, tumors with low or undetectable levels of the receptor would represent the primary targets against which to test ST1926 in phase II clinical trials. Finally, the genome-wide microarray data are a useful resource to design rational combinations of RRMs and other chemotherapeutics.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: ATRA, all-trans-retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RRM, retinoid-related molecule; CD437, (E)-3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid; ST1926, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid; BAPTA, 1,2 bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester); FURA-2, Fura-2 acetoxymethyl ester; DEVD-amc, N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin; PBS, phosphate-buffered saline; PI, propidium iodide; RT-PCR, reverse transcription-polymerase chain reaction; DMSO, dimethyl sulfoxide; ANOVA, analysis of variance; WT, wild-type; FACS, fluorescence-activated cell sorting; TNF, tumor necrosis factor; AV, annexin V.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Enrico Garattini, Laboratory of Molecular Biology, Centro Catullo e Daniela Borgomainerio, Istituto di Ricerche Farmacologiche "Mario Negri", via Eritrea 62, 20157 Milano, Italy. E-mail: egarattini{at}marionegri.it
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F/min) and calculated during the linear phase of calcium rise. In the case of the results shown in C, before addition of RRMs, cells were preincubated in the presence or absence of 10 and 50 µM BAPTA for 2 min. Each experimental value is the mean ± S.D. of three replicate cell cultures. *, significantly higher than the relative control value (p < 0.01 according to the Student's t test). D, DEVD-amc hydrolytic activity was measured in extracts of cells treated for 24 h with ST1926 (1 µM) or CD437 (1 µM) in the presence or absence of BAPTA (10 µM). The results are expressed in fluorescence arbitrary units (A.U.) and represent the mean ± S.D. of three replicate cell cultures. *, significantly higher than the relative control value (p < 0.01 according to the Student's t test).
