Role of peroxisome proliferator-activated receptor-gamma and its coactivator DRIP205 in cellular responses to CDDO (RTA-401) in acute myelogenous leukemia

Abstract

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor (NR) family of transcription factors with important regulatory roles in cellular growth, differentiation, and apoptosis. Using proteomic analysis, we showed expression of PPARgamma protein in a series of 260 newly diagnosed primary acute myelogenous leukemia (AML) samples. Forced expression of PPARgamma enhanced the sensitivity of myeloid leukemic cells to apoptosis induced by PPARgamma agonists 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-(12,14)-15DPGJ(2), through preferential cleavage of caspase-8. No effects on cell cycle distribution or differentiation were noted, despite prominent induction of p21 in PPARgamma-transfected cells. In turn, antagonizing PPARgamma function by small interfering RNA or pharmacologic PPARgamma inhibitor significantly diminished apoptosis induction by CDDO. Overexpression of coactivator protein DRIP205 resulted in enhanced differentiation induction by CDDO in AML cells through PPARgamma activation. Studies with DRIP205 deletion constructs showed that the NR boxes of DRIP205 are not required for this coactivation. In a phase I clinical trial of CDDO (RTA-401) in leukemia, CDDO induced an increase in PPARgamma mRNA expression in six of nine patient samples; of those, induction of differentiation was documented in four patients and that of p21 in three patients, all expressing DRIP205 protein. In summary, these findings suggest that cellular levels of PPARgamma regulate induction of apoptosis via caspase-8 activation, whereas the coactivator DRIP205 is a determinant of induction of differentiation, in response to PPARgamma agonists in leukemic cells.

Fig. 1

A. RT-PCR of neomycin gene expression in pcDNA3 or wt-PPARγ transfected clones. B. PPARγ and FLAG protein expression in transfected cells was detected by immunoblotting. C. Leukemic cells transfected with pcDNA3 or wt-PPARγ were treated with 1 μM CDDO for 5 hours, and PPARγ protein levels were examined by immunocytochemistry. D. PPARγ transcript was measured in wt-PPARγ transfected cells (clone 1) or in the same cells transfected with scramble or PPARγ siRNA, by real-time PCR. Results are reported as the mean number of transcripts per hundred transcripts of ABL1. Error bars denote the standard error of the mean.

Fig. 2

A. U937( pcDNA3) and U937 (pcDNA3-wt-PPARγ) cells were cultured in the presence of CDDO or vehicle for 24h, and apoptosis induction was analyzed by Annexin V flow cytometry at 24 hours. B. U937( pcDNA3) and U937 (pcDNA3-wt-PPARγ) cells were grown in the presence of 1μM CDDO or vehicle for 24h. Expression of PARP, caspase-3,-8, -9, HO-1 and GRP78 was analyzed by immunoblotting, and effects of CDDO on DNA fragmentation were determined. C. Caspase-8, and -9 processing and DNA fragmentation at 5 hours and 24 hours was measured. D. U937-wt-PPARγ cells were transiently transfected with scramble or PPARγ siRNA (siRNA1 and siRNA2), at a final concentration of 67nM. Seventy-two hours after transfection, cells were exposed to indicated concentrations of CDDO for 24 hrs. Induction of apoptosis was measured by annexinV flow cytometry. The data represent average results from three independent experiments. Solid lines, paired t-test comparing apoptosis in scrambled siRNA-transfected cells with apoptosis in PPARγ siRNA transfectants (p<0.01); dashed lines, paired t-test comparing apoptosis in untransfected cells with PPARγ siRNA transfectants (p<0.001).

Fig. 3

A. U937( pcDNA3) and U937 (pcDNA3-wt-PPARγ) cells were cultured in the presence of 1μM CDDO or vehicle for 24h, and p21 transcript was measured by real-time PCR. B. Cells were harvested at 24h, and p21/p27 levels were determined by immunoblotting. C. The fold transactivation of the p21 promoter by CDDO (0.75μM, 72 hrs). SW480 colon cancer cells were co-transfected with the wt- or mutant −2325/+8 p21 promoter-luciferase reporter vectors. Cells were co-transfected with β-galactosidase for normalization of the transfection variability. The fold transactivation of each p21 reporter construct is shown on top of the graph. D. Analysis of p21 levels in wt-PPARγ clone 1 electroporated with scrambled or PPARγ siRNA, 48h after transfection. Following 48 hrs, DMSO or CDDO were added for the next 24 hrs, and after 24hrs p21, HO-1, GRP78 and β-actin levels were determined by quantitative TaqMan PCR and Western blot analyses.

Fig. 4

A. MCF-7 cells were transiently co-transfected with 1μg of pPPRE-TK-LUC and 4μg of pcDNA3 or pcDNA3-DRIP205, treated with indicated concentrations of CDDO or 15dPGJ₂ for 92.5 hours, after which PPARγ transactivation was determined by relative luciferase activity calculated by dividing luciferase activity by protein concentration for each well. Results are shown as mean ± SEM for three separate experiments. B. Map of GAL4-DRIP fusion proteins. C. Coactivation of PPARγ by GAL4-DRIP fusion proteins. SW480 cells were co-transfected with 1000 ng of pPPRE3-LUC, 250 ng of β-galactosidase, 500 ng of pM empty or pM DRIP deletion mutants, treated with DMSO and 0.75μM CDDO, and luciferase activity was determined. Results are shown as mean ± SEM for three separate experiments.

Fig. 5

A. Nuclear fractions of HL-60 cells tranfected with vector (pcDNA3) or pcDNA3-DRIP205 constructs were lysed and analyzed by western blot analysis. PCNA was used as a loading control for nuclear lysates. B, C. HL-60 cells tranfected with vector (pcDNA3) or pcDNA3-DRIP205 constructs were treated with 0.5 μ M CDDO for 120 hours, and induction of myelomonocytic differentiation was determined by CD11b flow cytometry (%CD11b(+) cells, (B)) or NBT assay (C). Data represent average results from three independent experiments. D. HL-60 cells tranfected with vector (pcDNA3) or pcDNA3-DRIP205 constructs were pretreated with 4 μ M T007, a PPARγ antagonist, for 1 hour, followed by 0.75 μ M CDDO for 120 hours. Data represent average results (mean±SD) of three independent experiments. Induction of differentiation was determined by CD11b flow cytometry. ** denotes p=0.01, and *p=0.02.

Fig. 6

A. Peripheral blood (PB) or bone marrow (BM) samples from patients enrolled in the Phase 1 clinical trial were lysed and probed with DRIP205 and PPARγ by Western blot. β-actin was used as a loading control. In baseline sample from patient#2, not enough material was available for immunblotting. B. Patients were treated with CDDO (RTA-401) during a Phase I clinical trial, and cells were collected from the peripheral blood (PB) or bone marrow (BM) and assessed for expression of surface markers CD11b, CD14, CD33 and CD34 by flow cytometry at the indicated times (see also suppl. Table 2). Four of nine patients (patients #301, 304, 305, 306) showed alterations of these parameters during the observed period. PB baseline percentages are not available from patient #301, therefore, BM percentages are provided. C. Cells from the PB or BM were counterstained with CD34-APC and TMRM. Three of five patients (patients #301, 303, 305) showed alterations of these parameters during the observed period. Data are presented as percentage of CD34+ cells that have lost mitochondrial membrane potential (TMRM-low). BM baseline percentages are not available from patient #305.