Retinoic acid (RA) is used to treat leukemia and other cancers through its ability to promote cancer cell differentiation. Strategies to enhance the anticancer effects of RA could deepen and broaden its beneficial therapeutic applications. In this study, we describe a receptor cross-talk system that addresses this issue. RA effects are mediated by RAR/RXR receptors that we show are modified by interactions with the aryl hydrocarbon receptor (AhR), a protein functioning both as a transcription factor and a ligand-dependent adaptor in an ubiquitin ligase complex. RAR/RXR and AhR pathways cross-talk at the levels of ligand–receptor and also receptor–promoter interactions. Here, we assessed the role of AhR during RA-induced differentiation and a hypothesized convergence at Oct4, a transcription factor believed to maintain stem cell characteristics. RA upregulated AhR and downregulated Oct4 during differentiation of HL-60 promyelocytic leukemia cells. AhR overexpression in stable transfectants downregulated Oct4 and also decreased ALDH1 activity, another stem cell–associated factor, enhancing RA-induced differentiation as indicated by cell differentiation markers associated with early (CD38 and CD11b) and late (neutrophilic respiratory burst) responses. AhR overexpression also increased levels of activated Raf1, which is known to help propel RA-induced differentiation. RNA interference-mediated knockdown of Oct4 enhanced RA-induced differentiation and G0 cell-cycle arrest relative to parental cells. Consistent with the hypothesized importance of Oct4 downregulation for differentiation, parental cells rendered resistant to RA by biweekly high RA exposure displayed elevated Oct4 levels that failed to be downregulated. Together, our results suggested that therapeutic effects of RA-induced leukemia differentiation depend on AhR and its ability to downregulate the stem cell factor Oct4. Cancer Res; 71(6); 2371–80. ©2011 AACR.
Because malignant cell transformation is often associated with a maturational block, mechanisms of overcoming the differentiation block have engendered therapeutic interest. Retinoids have been shown to induce differentiation and have antiproliferative activities against skin, head and neck, breast, uterine, cervical, and liver cancer, although the most effective activity is against acute promyelocytic leukemia (APL; refs. 1–3). Retinoic acid (RA) is known to induce cell differentiation through RAR/RXR nuclear receptor activation. Aryl hydrocarbon receptor (AhR) is another nuclear receptor with a proposed role in differentiation. Recently, AhR has been shown to propel breast cancer (4) and liver cancer (5) cell differentiation. AhR has been found to be expressed in all tissues analyzed. It is present in the cytosol and in the nuclei. Two AhR functions are known, both being ligand dependent. It is a basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) transcription factor (6) and also an adaptor in the cullin 4B ubiquitin ligase complex (7). The transcriptional activity is the most studied, especially in the regulation of detoxification enzymes such as CYP1A1 (8). The role in the ubiquitin complex is emerging and has been found to be important for estrogen receptor α and androgen receptor degradation (9).
It has been shown that a limited number of transcription factors are needed to induce the self-renewing pluripotent stem cell state (10–12). Yamanaka and coworkers proposed Oct4, SOX2, KLF4, and c-Myc (10), whereas Thomson and coworkers proposed Oct4, SOX2, NANOG, and Lin28 (11) as essential factors for inducing the self-renewal stem cell state. A number of subsequent publications showed that, under certain conditions, the number could be further reduced to Oct4, SOX2, NANOG (13), or only Oct4 (14). Thus, Oct4 is the only currently known essential regulator/inducer of induced pluripotent stem cells among the Yamanaka/Thomson factors. As such, Oct4 becomes a prominent candidate as a regulator of cell differentiation caused by the embryonic morphogen RA.
There are reasons to suspect that RAR/RXR-, AhR-, and Oct4-controlled pathways are interrelated. The RAR/RXR and AhR pathways are known to cross-talk. For example, these receptors compete for SMRT protein and are upregulated by the same chemicals (15). In the case of the estrogen receptor (ER), another nuclear receptor which can also form heterocomplexes with RXR, AhR binds to ER response elements in target gene promoters (16). AhR can also target ER for degradation, a manifestation of the AhR cytosolic function (7). There may ergo also be multiple levels of cross-talk between AhR and RAR /RXR. Some findings suggest that cross-talk between the AhR and RA pathways occurs during cell differentiation. Teratogenic effects such as cleft palate and hydronephrosis can be induced by retinoids (17) and also by an AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (18). In fish, RA and its receptors are required both for AhR transcription and for embryonic development of blood vessels and bones (19).
We conducted an in silica search for putative response elements in the Oct4 promoter/enhancer region, using Genomatix software. We have found very close putative response elements for AhR/ARNT (225 to 249+) and RAR/RXR (230 to 254+) in Oct4B 5′ sequences and also AhR/ARNT (82 to 106+) and RAR/RXR (108 to 132+) in Oct4A, the form with stem cell–promoting properties, 5′ sequences. There was also another AhR site in each promoter region and another 4 RXR and 7 RXR sites for Oct4B and Oct4A, respectively. It is indeed known that the Oct4 promoter contains RA response elements responsible for Oct4 repression (20), but this might occur in cooperation with AhR in view of the sequence analysis, as well as with the coincidence of teratogenic effects driven by activating the RA or AhR pathways.
The present report shows that RA upregulates AhR expression and downregulates Oct4 when inducing the myeloid differentiation of HL-60 human leukemia cells. If these changes are of functional significance, we expect cells overexpressing AhR to differentiate faster than the parent cell line after RA treatment. Similarly, we expect cells expressing less Oct4 (Oct4 knockdown cells) to differentiate faster than their parent cell line. We created these stable transfectants and found that this was the case. AhR overexpression downregulated Oct4 and increased the amount of activated Raf1, which is known to drive RA-induced differentiation, and differentiation was enhanced. Differentiation was measured by the CD38 and CD11b cell surface markers and by the functional differentiation marker, inducible oxidative metabolism, namely, the respiratory burst characteristic of mature myeloid cells. Oct4 siRNA knockdown enhanced RA-induced differentiation, too. The results suggest that RA-induced differentiation depends on AhR and the mechanism involves downregulation of Oct4. The HL-60 cell line, a human myeloid (FAB M2) precursor cell capable of induced myeloid or monocytic differentiation, was used as an experimental system for RA-induced myeloid differentiation. It is an archetype model for the mechanism of action of RA that has been highly studied with many specific features of the RA-induced differentiation process very well defined. It is also a PML-RARα–negative system still responsive to RA and therefore it has the potential to highlight pathways involved in RA-induced differentiation that might be exploited in cancers other than APL.
Materials and Methods
Cell culture and treatments
HL-60 human myeloblastic leukemia cells were grown in RPMI 1640 supplemented with 5% heat-inactivated FBS (both from Invitrogen) and 1× antibiotic/antimycotic (Sigma) in a 5% CO2 humidified atmosphere at 37°C. The cell lines were derived from the original isolates and were a generous gift of Dr. Robert Gallagher and maintained in this laboratory. RA-resistant cells (RH cells) were created by maintaining the cells in high-density cultures (above 2 × 106 cells/mL) and treating them with 9 μmol/L RA once every 2 weeks. For treatments, all-trans-RA; Sigma) was added from a 0.5 mmol/L stock solution in ethanol with a final concentration of 1 μmol/L in culture. Valproic acid (VPA) was added to a final concentration of 1 mmol/L. The VPA treatment was 4 hours before the RA treatment, and all the times of indicated endpoints are with respect to RA treatment as the start. Experimental cultures were initiated at a density of 0.1 × 106 cells/mL. The RH cells were kept at normal density and no RA for a week before the experiments. Viability was monitored by 0.2% trypan blue (Invitrogen) exclusion and routinely exceeded 95%. All reagents were purchased from Sigma unless otherwise mentioned.
The AhR overexpressor in pCMV6-XL4 vector was obtained from OriGene. The Oct4 siRNA in U6.1/Neo was purchased from GenScript. For transfection in HL-60, 50-μg plasmid and 50-μL Lipofectamine 2000CD (Invitrogen) were incubated in 450 μL serum-free RPMI on ice for 15 minutes and then used to resuspend 10 × 106 cells and electroporated immediately at 300 mV.
CD38, CD11b quantification
Expression of cell surface differentiation markers was quantified by flow cytometry. A total of 0.5 × 106 cells were collected from cultures and centrifuged at 1,000 rpm in a microfuge for 5 minutes. Cell pellets were resuspended in 200 μL 37°C PBS containing 2.5 μL of antibody, APC, or PE-conjugated CD11b or CD38, as indicated in the Results section (all from BD Biosciences). Following 1-hour incubation at 37°C, cell surface expression levels were analyzed by flow cytometry (BD LSRII flow cytometer; BD Biosciences). APC fluorescence is excited at 633 nm and collected with a 660/20 band-pass filter. PE fluorescence is excited at 488 nm and collected with a 576/26 band-pass filter. Undifferentiated control cells were used to determine the fluorescence intensity of cells negative for the respective surface antigen. The gate to determine percent increase of expression was set to exclude 95% of the control population.
Respiratory burst quantification
Respiratory burst, a functional differentiation marker, was measured by flow cytometry. A total of 1 × 106 cells were collected and centrifuged at 1,000 rpm for 5 minutes in a microfuge. Cell pellets were resuspended in 500 μL at 37°C PBS containing 5 μmol/L 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro–fluorescein diacetate acetyl ester (H2-DCF; Molecular Probes) and 0.2 μg/mL 12-o-tetradecanoylphorbol-13-acetate (TPA; Sigma). Both, H2-DCF and TPA stock solutions were made in DMSO at concentrations of 0.2 mg/mL and 5 mmol/L, respectively. A control group incubated in H2-DCF and DMSO only was included. Cells were incubated for 20 minutes at 37°C prior to analysis by flow cytometry (BD LSRII). Oxidized DCF was excited by a 488-nm laser and emission collected with a 530/30 nm band-pass filter. The shift in fluorescence intensity in response to TPA was used to determine the percentage of cells capable of inducible oxidative metabolism (21). Gates to determine percent positive cells were set to exclude 95% of control cells not stimulated with TPA.
A total of 1 × 106 cells were collected by centrifugation and resuspended in 200 μL of cold propidium iodide (PI) hypotonic staining solution containing 50 μg/mL propidium iodine, 1 μL/mL Triton X-100, and 1 mg/mL sodium citrate. Cells were incubated at room temperature for 1 hour and analyzed by flow cytometry (BD LSRII), using 488-nm excitation and emission collected with a 576/26 band-pass filter. Doublets were identified by a PI signal width versus area plot and excluded from the analysis (21, 22).
Nuclear Oct4 quantification
At total of 1 × 106 cells were collected from cultures and centrifuged at 1,000 rpm in a microfuge for 5 minutes, resuspended in 200 μL of cold PI hypotonic solution as previously described for nuclei isolation (23), incubated on ice for 15 minutes, centrifuged again at 1,000 rpm for 5 minutes, fixed by resuspension in 100 μL of PBS with 2% paraformaldehyde (PFM; Alfa Aesar), and incubated at room temperature for 10 minutes followed by addition of 900 μL of ice-cold methanol to obtain a 90% methanol solution. Following incubation for 1 hour at −20°C samples were washed 2 times in hypotonic solution and resuspended in 200 μL PI hypotonic solution containing 2.5 μL of Oct4 (Santa Cruz Biotechnology) primary antibody. Following a 4°C overnight incubation period, nuclei were washed once with 1 mL PI hypotonic solution and stained with secondary AlexaFluor 350 goat anti-rabbit antibody for 1 hour and then analyzed by flow cytometry (BD LSRII) after 1 wash in PBS. Excitation was at 325 nm and emission was collected with a 440/40 band-pass filter. Nuclei unstained with the primary antibody but incubated with the secondary antibody were used to generate the background signal. Logic gates were set to include all the nuclei, and the results are given as the percentage of the mean fluorescence intensity of untreated nuclei.
Aldehyde dehydrogenase enzymatic activity assay
ALDH1 enzymatic activity was measured using the Aldefluor kit (Stem Cell Technologies) as described by Ginestier and coworkers (24), with the modification that the cells were incubated with the substrate at 37°C for 50 minutes instead of 40 minutes.
Protein detection by Western blotting
A total of 2 × 107 cells were lysed for total lysate, using 200-μL lysis buffer (Pierce) and lysates were cleared by centrifugation at 13,000 rpm for 20 minutes at 4°C after 3 cycles of freeze-thaw. For detection of nuclear proteins, 100 μL hypotonic lysis buffer was used to obtain the nuclei, which were then lysed in RIPA buffer (Sigma) as previously described (23). For AhR detection, 25 μg protein (total and nuclear lysates) was resolved on a 7.5% polyacrylamide gel. The same amount of protein was used on 12% gels for the other proteins. The electrotransfer was for 1 hour at 300 mA. The membranes were incubated with the indicated primary antibody at 4°C overnight. Anti-AhR antibody was from Santa Cruz Biotechnology. All the others were from Cell Signaling. Horseradish peroxidase–linked anti-mouse [for extracellular signal regulated kinase (ERK) 1/2] or anti-rabbit (for all others) IgG secondary antibodies (Cell Signaling Technology) and ECL (GE Healthcare) were used for detection. All blots were repeated 3 times.
Statistical analyses were done using SYSTAT 8.0 software. Means of treatment groups of interest were compared using the paired sample t test. All treatment groups were compared with control cells at the same time point. The data represent the means of 3 repeats ± SEM. A P value of <0.05 was considered significant.
RA-induced differentiation correlates with increased AhR levels and decreased Oct4 nuclear levels
RA treatment of HL-60 cells induces expression of a series of phenotypic markers consistent with induced differentiation. CD38 receptor expression can be detected within 6 hours of treatment, and 100% of the cells express it within 24 hours. CD38 is a type II transmembrane glycoprotein, expressed on several leukocytes and early hematopoietic precursor cells (25), that can signal through Raf and ERK activation (26). Expression of CD11b, an integrin receptor subunit that is also a marker for myeloid differentiation, occurs with slower kinetics than CD38 post–RA treatment. After expression of these markers, G0 cell-cycle arrest becomes apparent by approximately 72 hours of RA treatment. Similarly, inducible oxidative metabolism, a functional marker that is the antimicrobial respiratory burst of neutrophils, also finally becomes apparent (Fig. 1A). As RA induces this phenotypic conversion, AhR expression is upregulated without nuclear translocation (Fig. 1B) and nuclear Oct4 protein abundance is downregulated (Fig. 1C). The upper band on the AhR detected on the 7.5% gel may reflect phosphorylation-induced gel mobility retardation, as AhR is well known to be phosphorylated upon activation (27). In contrast to Oct4, Oct1 is not downregulated (Fig. 1D). We have previously shown that BLR1 receptor expression is essential for RA-induced differentiation toward neutrophils and that BLR1 expression depends on the transcriptional activity of Oct1 (28). Although Oct1 and Oct4 have structural and functional similarities, only Oct4 confers pluripotency (29), consistent with their differential regulation observed here. As expected for differentiating cells (24, 30), and consistent with previous reports showing that RA downregulates ALDH (31), we found that ALDH1 enzymatic activity is downregulated by 1 μmol/L RA treatment, assessed 48 hours posttreatment as cells undergo differentiation (Fig. 1E). In sum, RA-induced phenotypic conversion to a differentiated cell is associated with AhR upregulation and Oct4, but not Oct1, downregulation.
AhR upregulation enhances RA-induced differentiation, increases Raf pS621 phosphorylation, and decreases levels of nuclear Oct4
To determine whether RA-induced AhR expression has functional significance for differentiation, AhR-stable transfectants were created. Overexpression of AhR in stable transfectants enhances RA-induced differentiation. There is a significant increase of RA-induced CD38 and CD11b expression compared with parental cells (Fig. 2A and B). Inducible oxidative metabolism, the respiratory burst functional differentiation marker, is also enhanced (Fig. 2C). AhR expression by itself decreases nuclear Oct4 protein levels (Fig. 2D). Consistent with our hypothesis that AhR upregulation propels differentiation, ALDH1 activity is downregulated (P = 0.0079) in AhR overexpressors compared with wild-type (WT) cells (Fig. 2E). Figure 3A shows a Western blot confirming the overexpression of AhR in the AhR-stable transfectants [control WT HL-60 vs. control AhR-transfected HL-60]. AhR is well known to be phosphorylated upon activation (27), and resolution of the protein on a 7.5% gel shows phosphorylation-induced gel retardation consistent with this. AhR protein in AhR overexpressors, as in WT cells, did not translocate to the nucleus (Fig. 3B). AhR has a known cytosolic function acting as a ligand-dependent adaptor in the cullin 4B ubiquitin ligase, whereby it has been found to decrease ER expression and might decrease nuclear Oct4 levels in a similar way (7).
RA-induces mitogen activated protein kinase (MAPK) signaling which is necessary to propel differentiation (32, 33). The MAPK signaling utilizes Raf, activation of which is necessary to induce terminal myeloid differentiation (34). One might anticipate from the present results that AhR expression resulting in enhanced RA-induced CD38 expression would also result in enhanced Raf activation betrayed by S621 phosphorylation. Western blot analysis confirms that the AhR-transfected cells had enhanced Raf activation (Fig. 3A, control wt HL-60 vs. control AhR-transfected HL-60). Surprisingly, enhanced Raf activation due to AhR overexpression does not result in increased ERK phosphorylation compared with basal levels in parental WT controls. RA treatment has been found to cause phosphorylation of ERK1/2 (32, 33), which is confirmed here (Fig. 3C). But AhR overexpression suppresses that, suggesting a potential negative regulatory relationship between AhR and ERK. Interestingly, the reverse has been reported by Chen and coworkers (35), who showed that upon ERK kinase inhibition, AhR protein level accumulates. The results thus show that AhR expression enhances Raf activation, which is consistent with previously reported results on Raf propulsion of differentiation (34). They also suggest the conjecture that Raf can do this independent of activating ERK, possibly reflecting Raf functions other than activation of MEK (MAP/ERK kinase)/ERK such as its nuclear translocation (23) and association with nuclear proteins such as Rb (36), which is a target downregulated by RA (37, 38)
VPA, an HDAC inhibitor, is known to induce expression of cytochrome family members that are transcriptionally upregulated by AhR (39). One might thus anticipate that VPA could cause AhR expression and enhance RA-induced differentiation. Figure 3A confirms AhR upregulation by VPA, alone or in combination with RA. VPA causes enhanced Raf activation (Fig. 3A). However, as for AhR overexpression, VPA suppresses ERK activation in the absence or the presence of RA, although total ERK protein is upregulated (Fig. 3C). VPA thus causes AhR overexpression with corresponding signaling effects previously found associated with AhR upregulation.
The ability of VPA to upregulate AhR-motivated testing its effects on differentiation. VPA treatment induced the expression of the CD11b cell surface differentiation marker early after treatment (24 hours). RA-induced CD11b expression was also enhanced by cotreatment with VPA at 24 hours after treatment (Fig. 4A). VPA also caused downregulation of nuclear Oct4 expression (Fig. 4B and C). RA, AhR, and VPA thus all cause downregulation of Oct4, motivating the conjecture that Oct4 downregulation facilitates differentiation. Interestingly, all conditions achieved a similar amount of Oct4 downregulation, possibly reflecting tight control of Oct4 expression.
Decreased levels of nuclear Oct4 propel RA-induced differentiation
RA and increased AhR expression caused Oct4 downregulation. To determine whether Oct4 downregulation is functionally significant and facilitates RA-induced differentiation, Oct4 siRNA knockdown stable transfectants were created. The decreased nuclear Oct4 in stable transfectants is confirmed by flow cytometry (Fig. 5A) with a P value of 0.013. The knockdown achieved was comparable with the RA-induced reduction in expression. Although the Oct4 knockdown by itself does not precipitate differentiation, as evidenced by lack of CD38 or inducible respiratory burst, it does significantly (P < 0.05) enhance RA-induced CD38 expression, inducible oxidative metabolism (Fig. 5B and C), and increase in the percentage of cells in G1/0 (P = 0.015; Fig. 5D). It is noteworthy that the siRNA knockdown was demonstrable and statistically significant, but was modest; hence, even a relatively small apparent reduction in Oct4 can have demonstrable effects on facilitating RA-induced differentiation.
RA-resistant cells have increased levels of nuclear Oct4 after RA treatment
If Oct4 is essential for maintaining cellular stem-like properties—such as self-renewal and immature phenotype—and decreasing Oct4 levels facilitate RA-induced differentiation, then the failure of RA-resistant cells to differentiate may be associated with a failure to decrease expression of nuclear Oct4 after RA treatment. An RA-resistant derivative of the parental cells was created by subjecting cells maintained at high density to repeated high doses of RA. The resulting resistant cells grown from such treatment had lower levels of AhR that failed to be up regulated by RA (Fig. 6A). Although the RA-resistant cells have higher basal expression levels of CD38 before RA treatment than WT cells, RA treatment failed to increase expression levels of CD38 (Fig. 6B). RA-induced CD11b expression was also blocked in the resistant cells (Fig. 6C). Consistent with this, there is no RA-induced G0 block, and the G1/0, S, and G2 distribution is unaffected by RA treatment (Fig. 6D). The resistant cells had higher basal levels of Oct4 (P = 0.0043; Fig. 6E) and treated with RA failed to downregulate Oct4 levels. The significantly higher nuclear Oct4 levels in RA-resistant cells than in WT cells are consistent with the hypothesis that Oct4 downregulation promotes cell differentiation.
The present studies show that when RA induces myeloid differentiation of lineage uncommitted human myelomonocytic leukemia cells, HL-60, it causes upregulation of AhR and downregulation of nuclear Oct4. The functional significance of this was tested. Ectopic overexpression of AhR enhanced Raf activation, known to propel differentiation; downregulated Oct4, suggesting a downstream function of Oct4; and enhanced RA-induced differentiation indexed by cell surface markers and functional differentiation. Furthermore, VPA induced AhR upregulation and also enhanced RA-induced differentiation. Downregulation of Oct4 in siRNA stable transfectants also enhanced RA-induced differentiation, confirming the anticipated downstream role of Oct4 downregulation in facilitating differentiation. Interestingly, the modulation of AhR and Oct4 expression levels to have these effects apparently does not have to be large. Consistent with a suggested role for Oct4 downregulation in differentiation, RA-resistant HL-60 cells created by high-dose RA treatment had elevated Oct4 levels that did not downregulate. In sum, we have found that RA induces AhR upregulation and Oct4 downregulation, where AhR expression negatively regulates Oct4, and both occurrences promote RA-induced differentiation.
The finding that AhR overexpression and knockdown of Oct4 expression enhanced the induction of differentiation by RA is novel but consistent with other findings. Oct4 is known to support dedifferentiated stem cells (14, 29). RA, which induces differentiation, was reported to repress Oct4 protein expression (40), and we show here that AhR is involved in Oct4 downregulation by RA. RAR, RXR, AhR, and ER are members of nuclear receptor family of transcription factors binding as heterodimers and known for their cross-talk (7, 9, 15). In the case of AhR and ER, it has been shown that AhR can bind to ER response elements and augment ER responses (41). In HL-60 cells, estradiol at low doses stimulates proliferation and at high doses inhibits proliferation, similar to the dose–response effects of RA, suggesting the possibility of cross-talk between estrodiol and RA (42). Furthermore, estradiol/ER signaling has been found to enhance RA-induced HL-60 cell differentiation (43). Cross-talk between estrogen and RA has also been shown at the nuclear level, where for instance overlapping of ERE and RARE occurs as for the lactoferrin gene (44). ER could potentially thus be involved in relating AhR and RAR signaling and cross-talk. In the case of the Oct4A promoter, the AhR and RAR response elements are very close; hence, multiple ways of cross-talk could be possible, for example, by cooperation in protein–DNA binding, in recruiting cofactors, and inducing chromatin changes responsible for modulation of Oct4 gene expression. There are thus a number of reasons to anticipate that RAR, AhR, and Oct4 might be interrelated and that anticipation is borne out in the currently reported findings.
It has also been shown that AhR participates in ER and AR degradation by functioning as a ligand-dependent substrate-recognizing component of an ubiquitin ligase complex (7). Although it is beyond the scope of our present study to elucidate the mechanism of Oct4 downregulation by AhR, the fact that AhR was reported to downregulate other protein levels supports our report.
We found that AhR expression and its possible phosphorylation drive up Raf pS621 levels. Raf as part of the Raf/MEK/ERK MAPK signaling module has been found to be needed in RA-induced differentiation (34). ERK activation has been found with RA-induced differentiation. But the role of Raf, although important, is not fully mechanistically defined. We have previously shown that Raf pS621 accumulates in the nuclei during RA-induced differentiation and others have shown that AhR phosphorylation increases its nuclear activity. The fact that VPA increases Raf pS612 levels but inhibits occurrence of ERKpTEpY and that AhR overexpression due to ectopic expression also blunts RA-induced ERK activation suggests that ERK activation is not absolutely necessary. Of relevance, ERK has been found to be capable of acting as a scaffold to facilitate signaling without actual activation (45). The cumulative evidence points to the importance of Raf activation in contributing propulsion to RA-induced differentiation.
The current statistics associated with APL continue to show a relatively high recurrence rate together with a poor survival despite a very high rate of remission after the standard RA-induced differentiation therapy. The results presented here are of potential clinical significance. They show that in the standard RA-induced differentiation induction therapy the downregulation of stem cell–promoting factor, Oct4, is important in overcoming the maturation block and that this process is AhR dependent. Furthermore, VPA can promote aspects of this process.
Disclosure of Potential Conflicts of Interest
No conflicts of interest were disclosed.
This work was supported by grants from NIH [R01 CA033505 (A. Yen), U54 CA143876 (Craighead)], NYSTEM NY Department of Health. R.P. Bunaciu was supported in part by a Center for Vertebrate Genomics Scholarship.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received June 24, 2010.
- Revision received January 11, 2011.
- Accepted January 18, 2011.
- ©2011 American Association for Cancer Research.