From the X-Ceptor Therapeutics, Inc., San Diego, California 92121
Received for publication, March 3, 2003 , and in revised form, April 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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The clinical efficacy of 6-MP is due in part to antiproliferative and cytotoxic effects resulting primarily from the inhibition of purine biosynthesis at multiple steps and incorporation into nucleic acids as thioguanine nucleotides (27). More recent work has expanded the function of purine anti-metabolites by demonstrating that compounds such as 6-thioguanine or 6-mercaptopurine can target biological activities outside of the purine biosynthesis pathway including telomerase (5), protein kinase N (6, 7), axon growth and regeneration (8, 9), and apoptosis in B cells through the regulation of the Bcl-2/Bax ratio (10).
In this study, we have identified, from a high throughput screen, 6-mercaptopurine as a regulator of the transcriptional activity of the orphan nuclear hormone receptor Nurr1. There are three members of the NGFI-B group, including Nurr1 (NR4A2; RNR-1/HZF-3/TINUR/NOT), Nur77 (NR4A1; NGFIB/NAK-1/TR3/N10), and Nor-1 (NR4A3) (11, 12). The three receptors share extensive homology in the DNA binding domain (DBD) and the ligand-binding domain (LBD) but diverge significantly in the NH2 terminus. Mice lacking the Nurr1 gene have demonstrated a key role for this receptor in regulating the development of midbrain dopaminergic cells and controlling dopamine production through transcriptional regulation of tyrosine hydroxylase (1317). Through similar gene targeting experiments, a role for Nor-1 and Nur77 has been established in mediating T cell development through the control of apoptosis (18, 19). Additional functions for all three receptors have been identified in the regulation of genes that are part of the hypothalamic-pituitary-adrenal axis. Among these are the proopiomelanocortin (POMC) gene (2022) and the corticotropinreleasing hormone gene (23).
In order to identify potential small molecule regulators of Nurr1 and the other NGFI-B family members, a high throughput screening assay was developed and run on several compound libraries. The results presented here identify 6-mercaptopurine (6-MP) as a positive regulator of Nurr1 transcription activity. These data represent the first indication that 6-MP can regulate the transcriptional activity of a nuclear factor and implicate Nurr1 as a potential mediator of the antiproliferative effects of 6-MP. These results also validate the approach that small molecule compounds can be identified as regulators of orphan nuclear receptors independent of ligand binding.
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MATERIALS AND METHODS |
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Tissue Culture and TransfectionsCV-1 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) with 10% charcoal-stripped fetal calf serum (HyClone), 50 µg/ml gentamycin (Invitrogen) and plated at 10,000 cells/well in Dulbecco's modified Eagle's medium with 5% charcoal-stripped fetal calf serum (HyClone) and 50 µg/ml gentamycin into 96-well plates 1 day previous to transfecting. Transfections were carried out using FuGENE6 reagent (Roche Applied Science). Briefly, 70 ng of DNA were transfected per well with 0.25 µl of FuGENE6 reagent. For GAL4 plasmid transfections, 20 ng of MH100x4-tk-luciferase, 5 ng of the appropriate pCMX-GAL4-mNurr1 construct, 10 ng of pCMX-Gal, and 35 ng of pCMX as filler were transfected. For transfections using the POMC reporter, 30 ng of POMCx5-tk-luciferase, 2.5 ng of pCMX-mNurr1, 10 ng of pCMX-
Gal, and 27.5 ng of pCMX were used. For receptor selectivity assays, pCMX-mNor-1, pCMX-hFXR, pCMX-hRXR
, pCMX-hLXR
, pCMX-hROR
, and pCMX-hER
were used along with the different hormone response elements, POMCx5-tk-luciferase, ECREx7-tk-luciferase, CRBPIIx2-tk-luciferase, LXREx3-tk-luciferase,
-crystallin-HREx3-tk-luciferase, and EREx3-tk-luciferase, respectively. Compounds were added 5 h after transfection at the concentrations indicated in the figures, and the transfection was assayed 18 h later unless otherwise indicated. Cells were lysed, and the luciferase activity was measured using an LJL Analyst plate reader and normalized to
-galactosidase activity. All experiments were carried out a minimum of three times.
High Throughput ScreeningCompound assay plates were prepared by the addition of 0.5 µl of 1 mM stock solutions to each well of a 384-well plate. T175 flasks containing CV-1 cells were transfected with pCMX-Nurr1 along with POMCx5-tk-luciferase reporter using the FuGENE6 method described above. After 5 h, cells were trypsinized and replated into 384-well assay plates at 5000 cells/well and a final compound concentration of 10 µM. After overnight incubation, cells were lysed, and luciferase activity was determined by luminescence reading on the LJL Analyst plate reader. Data were visualized with the Spotfire software program.
PlasmidspCMX-MH100x4-tk-luciferase, pCMX-GAL, pCMXNurr1, pCMX-Nur77, pCMX-LXR
, pCMX-FXR, pCMX-RXR
, pCMXER
, and pCMX-ROR
were described previously (2428).
pCMX-Nor-1 was generated by subcloning of a cDNA fragment containing full-length human Nor-1 into the pCMX plasmid.
POMCx5-tk-Luc was generated by inserting five copies of an oligo-nucleotide (below) containing a Nurr homodimer response element (29) in front of a minimal thymidine kinase promoter driving expression of a luciferase gene: POMC NuRE, 5'-AGCTGATCGTGATATTTACCTCCAAATGCCAGATCGTGATATTTACCTCCAAATGCCAGATCGTGATATTTACCTCCAAATGCCA-3'.
The mNurr1 fragments, aa 1261, 143, 196, 1152, 1222, 44261, 96262, 152261, 223261, and 262583, were PCR-amplified and cloned to the TOPO vector (Invitrogen). They were sequencing-verified and subcloned to the pCMX-GAL4 vector. pCMX-mNurr1TAB1 was cloned by three-piece ligation of mNurr1-(183/84))
TAB1 fragment from TOPO-mNurr1-(185)
TAB1; mNurr1-(83/84598) fragment from pCMX-mNurr1; and pCMX vector at XhoI/SalI, BspEI, and NheI sites. The mNurr1-(185) fragment without TAB1 was PCR-amplified with mNurr1EXF and mNurr1
TAB1 primers and cloned to TOPO vector and sequencing-verified. The mNurr1
TAB1 primer has TAB1 deletion and a unique BspE1 site before the deletion.
Primers for PCR were as follows: mNurr1EXF, 5'-GAGCTAGTCGACTGTCCACCTTTAATTTCCTC-3'; mNurr1A261R, 5'-GCAAACAGCTAGCTAACCCTCATTGGAGGGAGAG-3'; mNurr1A262F, 5'-CCTCCAGTCGACGTCTGTGCGCTGTTTGCGG-3'; mNurr1A43R, 5'-GGTGAGCTAGCTAGCTAAACTTGACAAACTCTG-3'; mNurr1A44F, 5'-GAGTCAGTCGACGCATGGACCTCACCAACACTG-3'; mNurr1A96F, 5'-CAGAAGGTCGACAGATGCACAACTACCAGCAAC-3'; mNurr1A96R, 5'-CCATCGCTAGCTACTGAATGTCTTCTACCTTAA-3'; mNurr1A151R, 5'-GAGTTCGCTAGCTAGTTGTGAAGGGAGCCCGGA-3'; mNurr1A-152F, 5'-GCTCCGTCGACACTTCCACCAGAACTACGTGG-3'; mNurr1A222R, 5'-GGCTTGGCTAGCTAGTTGGGCACGGCGAAGGTCTG-3'; mNurr1A223F, 5'-TCGCCGGTCGACACCCCATTCGCAAGCCGGCA-3'; mNurr1 TAB1, 5'-GTCCGGACAGGGGCATGAAGCTGGGGAGAGAAGTGG-3'.
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RESULTS |
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6-Mercaptopurine Activation Is Specific for Nurr1 and Nor1The activity of 6-MP on Nurr1 was reconfirmed with resupplied stock of compound and demonstrated to be dose-responsive (Fig. 2A). 6-MP did not activate the reporter alone at the concentrations tested (not shown). Concurrent to the screen with Nurr1, a similar screen was developed for Nor-1. Upon testing of Nor-1 on the compound library, 6-MP was again identified as the sole positive hit. Nor-1 activation by 6-MP is similar in efficacy and potency to Nurr1 (Fig. 2B). These results suggested that 6-MP may regulate all Nurr members, so Nur77/NGFI-B was tested. Unlike Nurr1 and Nor-1, both the rat NGFI-B and human homologue TR3 were minimally activated by 6-MP (not shown). It is unclear if this is due to the much higher basal activity of Nur77/NGFI-B or if the 6-MP effect is limited to Nurr1 and Nor-1. It is possible that other factors may also influence activation of Nur77/NGFIB including cell type and/or reporter context. To establish whether the 6-MP effect was specific, several other nuclear hormone receptors were tested. LXR, FXR, RXR, ER, and ROR
showed no activation by 6-MP up to 50 µM (Fig. 2C). Although not exhaustive, these data suggest that the 6-MP effect on nuclear hormone receptors is limited to Nurr1 and Nor-1.
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6-Mercaptopurine Activates Nurr1 through the NH2-terminal AF-1Since their introduction as chemotherapeutic drugs in the late 1950s, 6-mercaptopurine and other purine anti-metabolites have been extensively studied both in the clinic and in vitro to identify their mechanism of action (see Refs. 2 and 3). There are several pathways in which 6-MP addition to cells may lead to activation of Nurr1, including binding as a ligand, inducing expression of endogenous Nurr1, perturbing purine biosynthesis, inducing apoptosis, or affecting other signal transduction pathways. To test the possibility that 6-MP is working as a ligand for Nurr receptors, constructs were generated with the LBD of Nurr1 and Nor-1 fused to the GAL4 DBD. In co-transfection experiments, these GAL4 fusions were not activated by 6-MP (Fig. 3A). To rule out the possibility that the 6-MP effect was through the LBD but required DNA binding to a hormone response element, GAL4 full-length Nurr1 was generated. The GAL4-Nurr1 was activated efficiently by 6-MP (Fig. 3A), suggesting that binding to the POMCx5 reporter did not contribute to the 6-MP activity. These results also demonstrate that the 6-MP effect is not due to induction of endogenous Nurr1 expression.
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Previous reports indicated that members of the NGFI-B family of nuclear receptors could be regulated by phosphorylation in the DNA binding domain, which led to an inhibition of DNA binding (3336). Additional work characterizing these receptors has demonstrated that, like other nuclear hormone receptors, the NH2 terminus contains a constitutive, ligand-independent transcription activation domain (AF1) that may also be a site for potential regulation (32, 36). To address the region of Nurr1 that is responsible for 6-MP activation, a series of GAL4-Nurr fusions were generated. The GAL4-Nurr1-(1598) contains the full-length Nurr1, whereas the other constructs are deletions of the region of Nurr1 that is NH2-terminal to the DNA binding domain (amino acids 1261). Western blot analysis confirmed the expression of these fusion proteins (not shown). Testing of these constructs demonstrated that 6-MP was activating through the NH2-terminal 261 amino acids (Fig. 3B). Since this region does not contain the DBD of Nurr1, these results rule out regulation of DNA binding or activation of the LBD as the mechanism for the 6-MP effect on Nurr1 and indicate that the AF1 may be the target of 6-MP. Additional analysis of the NH2 terminus with a larger set of GAL4-Nurr1 truncations revealed a minimal region between amino acids 1 and 151 that still retains the ability to respond to 6-MP (Fig. 3B), albeit reduced compared with the larger domain (aa 1222). The first 40 amino acids appear to be dispensable for activity, since the GAL4-Nurr1-(143) is inactive and GAL4-Nurr1-(44261) is active. To verify that 6-MP activation of Nurr1 is not restricted to CV-1 cells, the human embryonic kidney cell line 293 was transfected with GAL4-Nurr1-(1222). The 6-MP activation was comparable (Fig. 3C) with that observed in CV-1 cells, supporting the possibility that Nurr1 is a target for 6-MP in human tissues. A sequence comparison of the region 40151 between Nurr1 and Nor-1 indicates several stretches of conserved amino acid residues (Fig. 3D) and several potential phosphorylation sites. This region is consistent with the boundaries of the AF-1 domain characterized in previous studies and contains the TAB-1 activation region identified as a minimal activation region (32). In order to determine whether the TAB-1 was necessary for the 6-MP effect, a deletion mutant lacking TAB-1 was generated (Fig. 3E). Although the basal level of activity of the receptor was diminished, the magnitude of the 6-MP activation was not impaired, indicating the presence of additional novel regulatory regions in the receptor NH2 terminus.
In order to demonstrate that this region of Nurr1 was functioning as a substrate for a cell-based activity, co-transfection experiments were done to determine whether the NH2-terminal 261 amino acids could compete with both the constitutive activity of the full-length Nurr1 and 6-MP-activated full-length Nurr1. The ability of GAL4-Nurr1-(1261) but not GAL4-DBD to significantly inhibit the constitutive activity of full-length Nurr1 (Fig. 3F) indicates that either cellular co-factors or enzyme activities are required for Nurr1 activation through the AF-1. Work from others demonstrating interaction of this region with the p160 co-activator complex supports these results (37). The inhibition of 6-MP activation of Nurr1 (Fig. 3F) by the GAL4-Nurr1-(1261) suggests that these factors are potentially sensitive to regulation by components of the purine biosynthetic pathway.
Recent work has shown that signal transduction pathways such as protein kinase A and mitogen-activated protein kinase activation can regulate the AF-1 region of Nurr1 (38). Testing with compounds including SB-203580 (mitogen-activated protein kinase inhibitor), LY-294002 (phosphatidylinositol 3-kinase inhibitor), DRB (protein kinase A inhibitor), PD-98059 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor), and staurosporine (protein kinase C inhibitor), has not resulted in effects specific to either the constitutive activity of the Nurr1 NH2 terminus or to the 6-MP activation of this region (not shown). Since protein kinase A has been implicated in regulation of Nurr family members, we further tested two other protein kinase A inhibitors, 4-cyano-3-methylisoquinoline and KT5720, in their ability to influence 6-MP activation of Nurr1. These compounds also had no effect on either the basal activity of Nurr1 or the activation by 6-MP (not shown).
Activation of Nurr1 Is Specific to 6-Mercaptopurine and Not Other Compounds with Similar Mechanism of ActionThe majority of known functions of 6-MP involve conversion into the active metabolite 6-thio-IMP through the enzyme hypoxanthine-guanine phosphoribosyl transferase (39). At this point, the 6-thio-IMP can be utilized in two different pathways. One option is that the 6-thio-IMP can be methylated by thiopurine methyltransferase to 6-Me-thio-IMP, which is a potent inhibitor of de novo purine biosynthesis. The 6-Me-thio-IMP can also be derived from the intermediate 6-methylmercaptopurine riboside, which is converted to 6-Me-thio-IMP by adenosine kinase (39). The other option is that 6-thio-IMP can be metabolized into 6-thioguanosine 5'-monophosphate, which is then incorporated into DNA and RNA as 6-thio-GTP, leading to eventual cell death (40, 41). To determine whether either of these metabolic pathways were involved in the activation of Nurr1, a number of nucleotide analogs and compounds known to effect purine biosynthesis were tested. As mentioned previously, the compound library that was screened contains several other thiopurine compounds and nucleotides including thioguanine, adenosine, allopurinol, azathioprine, adenosine phosphate, and puromycin. Several of these compounds were resupplied and retested in both the full-length Nurr1 assay and the GAL4-Nurr1 assay. Adenosine, adenine, 6-TG, purine, hypoxanthine, and inosine were inactive on all Nurr1 constructs tested (Fig. 4A). Additional testing of other thiopurine compounds included 6-thioguanosine, 6-mercaptopurine riboside, 6-mercaptopurine deoxyriboside, 6-methyl-mercaptopurine (6-MMP), 6-methyl-mercaptopurine riboside (6-MMPR), and 2-mercaptopurine. Among these, only 6-mercaptopurine riboside and 6-mercaptopurine deoxyriboside were able to activate Nurr1 (Fig. 4B). This activity was also observed when assayed on the full-length receptor on the POMCx5 reporter (Fig. 4C). The inhibitory effects of 6-TG and 6-thioguanosine cannot be interpreted due to nonspecific inhibition of both the POMCx5 reporter as well as the MH100x4 reporter.
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The finding that 6-MMP and 6-MMPR did not activate Nurr1 suggests that either there is not a sufficient amount of adenosine kinase required to convert these compounds to 6-Me-thio-IMP (39) or 6-MP, 6-MMP, and 6-MMPR inhibit purine de novo biosynthesis through slightly different mechanisms. To address the possibility that adenosine kinase activity is required for activation of Nurr1 by 6-MP, we tested 6-MP in the presence of the adenosine kinase inhibitor 5'-iodotubercidin. The activation of full-length Nurr1 by 6-MP is completely inhibited in a dose-dependent manner by 5'-iodotubercidin (Fig. 4D), suggesting that there is sufficient adenosine kinase activity in the cell to convert 6-MP to an active form and that this conversion is required for activation of Nurr1 by 6-MP. These results were also seen on the GAL4-Nurr1 full-length and GAL4-Nurr1-(1261) protein (not shown). Based on the known consequence of inhibiting adenosine kinase, there are two possible explanations for why 5'-iodotubercidin might inhibit 6-MP activity. The first is that, if 6-MP is converted to its metabolite 6-methyl-MP-riboside, then adenosine kinase is required for phosphorylation of this substrate to convert it to the 6-Me-IMP active metabolite. The second is that since adenosine kinase is the key enzyme in regulating the conversion of adenosine to AMP, inhibiting this step results in a build up of adenosine, which we have previously shown can inhibit 6-MP activation of Nurr1. This does not provide an explanation for why 6-MMP and 6-MMPR do not activate Nurr1. It is possible that these compounds are not cell-permeable and that exogenous 6-MMP and 6-MMPR may require additional modifications to be active.
6-Mercaptopurine Activation of Nurr1 Can Be Blocked by Replenishing Endogenous PurinesThe antiproliferative activity of 6-MP has been shown to result partly from a decrease in the production of adenosine and guanosine synthesis (42). To determine if depletion of these nucleotides had any effect on Nurr1, 6-MP was added in the presence of adenine, adenosine, guanine, guanosine, hypoxanthine, or inosine. Of these compounds, adenine, adenosine, guanosine, hypoxanthine, and inosine effectively inhibited the activation of Nurr1 by 6-MP both in the case of the GAL4-Nurr1 constructs (Fig. 4E) as well as full-length Nurr1 on the POMCx5 reporter (not shown). The inability of guanine to inhibit the 6-MP activation may be due either to solubility of the compound or a lack of conversion of guanine to the nucleoside form. These data indicate that the level of adenine, adenosine, or guanosine molecules can specifically affect the activity of Nurr1 through a region in the NH2 terminus. One consequence of reduced adenosine molecules is a reduction in the amount of AMP/ADP/ATP available to carry out cellular functions. This would imply that there exists the possibility of an ATP-dependent pathway that can regulate Nurr1 (and Nor-1) activity and that perturbation of this pathway results in the observed transcriptional activation detected in the presence of 6-MP. Indeed, recent reports suggest that a cAMP-dependent protein kinase A signaling cascade can regulate both Nurr1 expression as well as transcription activity (38).
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DISCUSSION |
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The activity of 6-MP was identified through a high throughput screen using a Nurr1 homodimer response element and full-length receptor. The initial screening result was confirmed on both full-length receptor and a GAL4 fusion, indicating that the 6-MP activity was not a result of influencing DNA binding to the POMC response element or a transcription artifact picked up during screening. Surprisingly, the 6-MP effect was not dependent on the ligand-binding domain, and the minimal response region was mapped to amino acids 40151 in the NH2 terminus (32). Whereas this region has previously been shown to contain a defined activation domain, TAB-1, our results indicate that it is not the target of 6-MP regulation. Testing on other nuclear hormone receptors demonstrates that the effect of 6-MP is selective toward members of the NGFI-B family of proteins. The specificity of the activity is striking when considered in the context of the broad consequences of 6-MP on purine biosynthesis and cell proliferation and further suggests that these receptors may play a distinct role in mediating 6-MP action in vivo. The results demonstrating that other purine anti-metabolites that are similar in structure and function to 6-MP, such as 6-thioguanine, azathioprine, and 6-MMPR, cannot activate Nurr1 support our finding as a novel and unique property of 6-MP.
Based on the current understanding of how 6-MP regulates certain cell functions (2, 3), there are several possibilities to explain the activation of Nurr1 and Nor-1. It has been demonstrated that 6-MP is processed into the active metabolite 6-thioinosine monophosphate through a series of enzyme conversions. The 6-thioinosine monophosphate can exert antiproliferative effects through conversion into thioguanine nucleotides that are incorporated into nucleic acids or through inhibition of the purine de novo biosynthetic pathway as 6-thioinosine monophosphate or a methylated form, Me-6-thioinosine monophosphate. In support of the latter possibility, our findings demonstrating that the addition of adenosine, guanosine, hypoxanthine, and inosine can block the activation of Nurr1 by 6-MP are consistent with previous studies showing that the cytotoxic effects of 6-MP can be reversed or inhibited by replenishing the pool of nucleotides through the addition of adenosine or guanosine (42). The activation of Nurr1 in CV-1 cells and other cell lines tested is observed at 10 µM 6-MP and increases with increasing concentrations of 6-MP. The range of concentrations of 6-MP needed to inhibit purine biosynthesis varies greatly from cell to cell, with sensitive cell lines such as the human lymphoblastic Molt F4 requiring 0.510 µM and some cell lines being resistant at concentrations above 100 µM. Therefore, whereas more potent derivatives of 6-MP may exist, most biological effects of 6-MP observed to date occur in the micromolar range, and activation of Nurr1 falls within this range. At this time, there is no evidence to suggest that the activation of Nurr1 by 6-MP is through the induction of cytotoxicity as a result of incorporation into DNA or RNA and subsequent DNA damage. The transfection efficiency as measured by constitutive expression of a -galactosidase gene is not affected by the addition of 6-MP. Further, other cytotoxic and antineoplastic compounds tested in the high throughput screen, such as carboplatin, melphalan, methotrexate, and the closely related purine analogs thioguanine and azathioprine, did not activate Nurr1. It is possible, however, that inhibition of DNA or RNA replication induces signaling cascades unique to 6-MP that result in the activation of Nurr1, although the relatively short exposure (overnight) to 6-MP would suggest that this is not the mechanistic pathway.
Purine anti-metabolites have also been shown to have other effects in the cell. Most pertinent to the observations presented in this report is the finding that 6-methyl-mercaptopurine riboside and 6-thioguanine are specific and effective inhibitors of protein kinase N, which has been implicated in nerve growth factor signaling (6, 7). Recent reports also suggest that 6-MMPR can regulate tyrosine kinase signaling in the process of angiogenesis (43). The precedent set by these findings suggests that 6-MP may also regulate as yet unidentified activities in the cell that may specifically influence Nurr1 activity. NGFI-B receptors, like many other nuclear hormone receptors, can be regulated through phosphorylation. Regulatory sites have been identified in the DNA binding domain and the NH2 terminus (3436, 38). Therefore, one potential mechanism for activation of Nurr1 may be through 6-MP regulation of a kinase or phosphatase that affects phosphorylation within the region of amino acids 44151. This region is rich in serines, threonines, and tyrosines. Consequences of the phosphorylation state could include interaction with co-factors and also regulation of intramolecular interactions. Recent work indicates that the region identified as responsive to 6-MP regulation is also the site of protein-protein interactions in Nur77 with transcriptional co-activators such as p300 and the p160/steroid receptor coactivator (37). Whereas Nur77 was not activated by 6-MP in our experiments, it is possible that under other conditions, activation may be seen. It is likely that the effect of 6-mercaptopurine will also depend on such factors as cell proliferation, availability of co-factors, DNA response element, and potentially conditions in which the receptors are overexpressed (i.e. immediate early response signaling or disease states).
The ability of 6-MP to regulate the activity of a transcription factor, such as Nurr1 or Nor-1, broadens significantly the possible mechanisms of action of this drug. Members of the NGFI-B family of nuclear receptors are expressed in lymphocytes, various cancer cells, and cells involved in inflammatory diseases, indicating that this group of receptors is present in the tissues targeted by 6-MP (12). Whereas many of the steps involved in 6-MP-mediated cytotoxicity have been elucidated, it is possible that these effects can be enhanced or suppressed through secondary targets. Nur77 and Nor-1 have been shown to play a role in mediating apoptosis in T lymphocytes in vivo and numerous cancerous cell lines (18, 19, 44, 45). When considering the question of whether regulation of apoptosis by Nur77/Nor-1 is through transcription regulation or other mechanisms, it is interesting to speculate that the effectiveness of 6-MP treatment may be influenced by the levels of these receptors.
The primary significance of the findings presented here is that there exists in the cell a signaling pathway that is sensitive to either levels of purine nucleotides or to direct regulation by 6-MP and that this pathway is specific toward the nuclear hormone receptors Nurr1 and Nor-1. The identification of 6-MP as a regulator of Nurr1 transcription introduces this compound as a tool for understanding the nature of the components of this pathway. It is expected that, upon further characterization of this signaling cascade, it should be possible to design pharmacological tools for the selective therapeutic regulation of Nurr1 receptors in treatment of central nervous system disorders including Parkinson's disease and inflammatory disorders. Additionally, the results presented here highlight a novel aspect of the potential mechanism of action of 6-MP, one of the most widely used antileukemic and anti-inflammatory drugs.
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FOOTNOTES |
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To whom correspondence should be addressed: X-Ceptor Therapeutics, Inc., 4757 Nexus Center Dr., San Diego, CA 92121. Tel.: 858-458-4553; Fax: 858-458-4501; E-mail: pordentlich{at}x-ceptor.com.
1 The abbreviations used are: 6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine; DBD, DNA binding domain; LBD, ligand-binding domain; POMC, pro-opiomelanocortin; 6-MMP, 6-methyl-mercaptopurine; 6-MMPR, 6-methyl-mercaptopurine riboside.
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ACKNOWLEDGMENTS |
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REFERENCES |
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