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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kogan, S. C. Articles by Bishop, J. M. Search for Related Content PubMed PubMed Citation Articles by Kogan, S. C. Articles by Bishop, J. M. Related Collections Plenary Papers Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 95 No. 5 (March 1), 2000: pp. 1541-1550 PLENARY PAPER Leukemia initiated by PMLRAR: the PML domain plays a critical role while retinoic acid-mediated transactivation is dispensable Scott C. Kogan, Suk-hyun Hong, David B. Shultz, Martin L. Privalsky, and J. Michael Bishop From the G.W. Hooper Foundation and Departments of Laboratory Medicine and Microbiology & Immunology, University of California, San Francisco, CA; and the Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA.     Abstract Top Abstract Introduction Materials and methods Results Discussion References The most common chromosomal translocation in acute promyelocytic leukemia (APL), t15;17(q22;q21), creates PMLRAR and RARPML fusion genes. We previously developed a mouse model of APL by expressing PMLRAR in murine myeloid cells. In order to examine the mechanisms by which PMLRAR can initiate leukemia, we have now generated transgenic mice expressing PMLRARm4 and RARm4, proteins that are unable to activate transcription in response to retinoic acid. PMLRARm4 transgenic mice developed myeloid leukemia, demonstrating that transcriptional activation by PMLRAR is not required for leukemic transformation. The characteristics of the leukemias arising in the PMLRARm4 transgenic mice varied from those previously observed in our PMLRAR transgenic mice, indicating that ligand responsiveness may influence the phenotype of the leukemic cells. The leukemias that arose in PMLRARm4 transgenic mice did not differentiate in response to retinoic acid therapy. This result supports the hypothesis that a major therapeutic effect of retinoic acid is mediated directly through the PMLRAR protein. However, a variable effect on survival suggested that this agent may be of some benefit in APL even when leukemic cells are resistant to its differentiative effects. Transgenic mice expressing high levels of RARm4 have not developed leukemia, providing evidence that the PML domain of PMLRAR plays a specific and critical role in the pathogenesis of APL. (Blood. 2000;95:1541-1550) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Retinoids are signaling molecules with significant roles in development and differentiation.1,2 These biologic effects led to the hypothesis that retinoids might be useful agents in the treatment of human malignancies. Hence, retinoids have been evaluated as possible therapies for a variety of human neoplasms including leukemias, skin cancers, cervical cancer, and neuroblastomas.3,4 Among myeloid leukemias, acute promyelocytic leukemia (APL) was found to be particularly sensitive to retinoic acid5 and more than a decade has passed since the demonstration that all-trans retinoic acid (tRA) could induce remission in patients with APL by stimulating differentiation of the leukemic cells.6,7 Understanding the pathogenesis and retinoid responsiveness of APL is important for expanding the application of retinoids in cancer treatment and for developing additional differentiation therapies. In 1977 Rowley and colleagues8 described a specific association of APL with a t(15;17) chromosomal translocation. Subsequent to the demonstration of the therapeutic benefit of tRA in APL, the breakpoint on chromosome 17 was identified to be within a gene encoding a retinoic acid receptor, RAR.9-12 The breakpoint on chromosome 15 was identified to be within a novel gene, PML.13-17 Expression of the PMLRAR fusion is a consistent feature of the disease in the vast majority of APL patients.18 Since these discoveries, efforts have been directed at understanding the role of PMLRAR in leukemogenesis and response to therapy. In addition to the common t(15;17) translocation, other chromosomal translocations have been identified in rare cases of APL. These translocations also result in fusions to RAR and include fusions with PLZF in t(11;17)(q23;q21),19 NPM in t(5;17)(q32;q21),20 and NuMA in t(11;17)(q13;q21).21 The partners of RAR in the APL fusions are all nuclear but otherwise have limited commonality. This fact raises the possibility that all the translocations contribute to APL pathogenesis by generating abnormal retinoic acid receptors that share common transcriptional properties. RAR is a ligand inducible transcription factor. In the absence of its ligand, tRA, RAR generally acts as a transcriptional repressor by recruiting corepressor molecules, including SMRT and N-CoR, which in turn recruit histone deacetylases. In the presence of ligand, RAR generally acts to induce transcription by releasing corepressor molecules and recruiting coactivators.22 When compared with RAR, PMLRAR has context dependent effects on transcription.13,15,17,23-25 For example, depending on cell type and the transcriptional element assayed, PMLRAR can decrease or increase basal transcription in the absence of ligand. Similarly, PMLRAR can exhibit both dominant negative activity and superactivation in the presence of tRA. Whether transcriptional activation and/or transcriptional repression by PMLRAR is necessary or sufficient for leukemogenesis has not been experimentally addressed. Although almost all APL patients respond to tRA therapy, resistance to this agent often develops in patients so treated.26,27 It has been suggested that enhanced metabolism of tRA, increased expression of the cellular retinoic acid binding protein II, and increased expression of the multidrug resistance gene product may contribute to clinical tRA resistance (reviewed in Ding et al28 and Imaizumi et al29). However, alterations of the PMLRAR protein itself have been described in some retinoic acid resistant subclones of the NB4 APL cell line, as well as in some patients with a disease that was clinically resistant to tRA.28-31 The observed mutations in PMLRAR included amino acid changes that impair the ability of the protein to bind retinoic acid and to activate transcription. These findings provided evidence that loss of ligand responsiveness by PMLRAR can play a role in clinical tRA resistance. We previously developed a murine myeloid leukemia model that recapitulates many of the features of APL.32 We have now generated additional transgenic mice to assess the role of hormone responsiveness by PMLRAR in both leukemogenesis and tRA response, as well as the sufficiency of transcriptional repression at retinoic acid response elements (RAREs) in initiation of leukemia.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Preparation of plasmid constructs The m4 mutation was introduced into the PMLRAR and RAR open-reading frames by a 2-step polymerase chain reaction (PCR) mutagenesis protocol, using 2 mutagenic primers, 5'-GATCACGCCGAAGATGGAGATCCC-3' and 5'-ATCTTCGGC GTGATCACCCGCTC-3'. The resulting PCR-generated fragment was digested with Bcl I and Xba I and was transferred into the pSG5-RAR or pSG5-PMLRAR background. The results of mutagenesis were verified by sequencing. A pGEX-KG construct was used to express either glutathione S-transferase (GST) or a GST-SMRT fusion (encoding codons 751-1495 of human SMRT) in Escherichia coli.33 Protease resistance assay 35S-radiolabeled PMLRAR and PMLRARm4 mutant proteins were synthesized in vitro by the TnT procedure. For the trypsin assay, 1 µL aliquots of in vitro translation product were diluted to a final volume of 20 µL each with 50 mmol/L Tris-Cl (pH 7.4) containing either tRA or an equivalent volume of ethanol carrier. Proteolysis was initiated by adding from 0 to 8 µgs of trypsin-TPCK per sample (trypsin pretreated with tosyl-L-phenylalanine chloromethyl ketone). The samples were then incubated at room temperature for 10 minutes. The proteolysis was terminated by addition of 14 µL of 5 × denaturing polyacrylamide gel electrophoresis (PAGE) sample buffer and the samples were rapidly frozen on dry ice. The samples were subsequently thawed, boiled for 10 minutes, resolved by denaturing PAGE, and visualized by autoradiography. Transient transfections CV-1 cell transfections were performed by a lipofection method as recommended by the manufacturer (Lipofectin, Gibco-BRL). Approximately 7 × 104 cells were transfected with 25 ng of the pSG5-RAR or pSG5-PMLRAR plasmids (representing "wild-type" or the m4 mutant), 100 ng of pCMV-lacZ (used as an internal normalization control for the efficiency of the transfection procedure) and 100 ng of the ptk-luciferase-RARE reporter. Five hours after transfection, the cells were transferred into media either lacking or containing 1 µmol tRA. Cells were harvested 48 hours after transfection and the levels of luciferase and -galactosidase were determined.34,35 In vitro receptor/corepressor binding assays GST-fusion proteins were expressed in E coli and were purified and immobilized by binding to glutathione agarose as previously described.34 35S-methionine-labeled full-length RAR, RARm4, PMLRAR, and PMLRARm4 proteins were synthesized by a coupled in vitro transcription and translation system (Promega TnT kit, Promega, Madison, WI). The radiolabeled proteins were subsequently incubated with the immobilized GST fusion proteins in HEMG buffer in the presence or absence of tRA, the agarose matrix was extensively washed and bound proteins were eluted with free glutathione and analyzed by denaturing PAGE.33 The electrophoretograms were visualized and quantified by phosphorimager analysis (Molecular Dynamics STORM system, Molecular Dynamics, Sunnyvale, CA). Generation of transgenic mice The human PMLRARm4 and RARm4 cDNAs were cloned into the hMRP8 expression cassette.36 Transgenic animals were prepared following standard procedures37 from inbred FVB/N mice.38 Western blotting and immunofluorescence Western blotting was performed as previously described with a rabbit polyclonal antiserum raised against a GST-fusion protein, encompassing amino acids 420-462 of the human RAR protein (anti-RARF).32,39 Whole-cell lysates of bone marrow from control and transgenic mice were subjected to denaturing PAGE on 8% or 12% SDS-polyacrylamide gels and were transferred to nitrocellulose. Immunofluorescence analysis of bone marrow cells was performed essentially as described40 but using the anti-RARF antiserum at a 1:150 dilution. Isolation of cells from tissues, cell staining, and fluorescence-activated cell sorting These were performed as previously described.32,40 In addition, Sudan Black B staining was performed using reagents from Sigma, according the manufacturer's directions. Peripheral blood counts Blood was analyzed on a Hemavet veterinary hematology analyzer to assess white blood cell counts, hemoglobin, and platelet counts. White blood cell differential counts were performed on peripheral blood smears. Methylcellulose cultures Bone marrow cells were cultured in duplicate in 35 mm petri dishes in Methocult M3230 methylcellulose medium (StemCell Technologies, Vancouver, BC) supplemented with either 50 units/mL G-CSF (Boehringer Mannheim), or 2.5 ng/mL GM-CSF (StemCell Technologies) plus 2% Xg63Ag8-653-IL341 conditioned medium. One milliliter cultures contained 5 × 104 viable bone marrow cells. Analysis was as previously described.40 Transplantations Cells isolated from bone marrow and spleens of leukemic animals were resuspended in buffered saline and injected into the tail veins of 6- to 12-week-old FVB/N mice, 5 × 106 viable cells/recipient. Nonleukemic bone marrow isolated from PMLRARm4 transgenic founder #4048 was transplanted into lethally irradiated FVB/N mice as previously described.32 Treatment with all-trans retinoic acid Leukemic mice were treated by subcutaneous implantation of 21-day release pellets containing 5 mg tRA or placebo (Innovative Research of America). Morphologic differentiation by tRA was assessed on days 4 and 11 of therapy.     Results Top Abstract Introduction Materials and methods Results Discussion References Generation of transgenic mice We generated transgenic mice expressing a PMLRAR unable to activate transcription as well as transgenic mice expressing an RAR with dominant negative activity. For this purpose, we introduced a Leu to Pro mutation at amino acid 398 of RAR into cDNAs encoding PMLRAR and RAR (Figure 1A). This mutation was originally identified by Shao and colleagues31 in a retinoic acid resistant subclone of human APL cells (NB4-R4) and was designated the m4 mutation. The m4 mutation impairs ligand binding, abrogates ligand-induced transcriptional activation, and blocks ligand-induced release of SMRT corepressor.31,42 Furthermore, PMLRARm4 and RARm4 act as dominant negative inhibitors of tRA-induced transcription.31 View larger version (27842135K): [in this window] [in a new window]   Fig 1. PMLRARm4 and RARm4. A, PMLRARm4: the Leu to Pro point mutation at codon 398 of RAR was introduced into a human PMLRAR cDNA whose chromosome 15 breakpoint lies in breakpoint cluster 1.16 The PML portion of the fusion is shown with hatching with selected structural domains labeled and shown in white. The B-F domains that encompass the RAR portion of the fusion are labeled and functional regions are noted. RARm4: the Leu to Pro point mutation was introduced into a human RAR1 cDNA. B, Hormone binding by PMLRAR ("Wild-type," WT) and PMLRARm4 (m4 mutant). Radiolabeled proteins synthesized by in vitro transcription and translation were incubated without or with increasing amounts of trypsin (indicated above the panels) in the absence or presence of 1 µmol tRA. The protein products were resolved by denaturing PAGE and visualized by autoradiography. The arrows show the position of the full-length undigested proteins. Smaller bands represent partially degraded products. C, Dominant negative activity of the m4 mutant proteins. CV-1 cells were transiently transfected with pSG5 constructs containing no exogenous receptor, RAR (WT), RARm4, PMLRAR (WT), and PMLRARm4. Luciferase activity expressed from a cotransfected RARE-luciferase reporter gene was normalized to -galactosidase activity from a cotransfected pCH210-LacZ plasmid. D, Decreased hormone-induced dissociation of SMRT corepressor by the m4 mutant proteins. GST-SMRT fusion protein was synthesized in E coli and was immobilized on glutathione agarose. The different receptor proteins were synthesized by in vitro transcription and translation and were incubated with the immobilized GST-SMRT in the absence or presence of 1 µmol tRA, as indicated below the panels. Equivalent amounts of GST-SMRT and radiolabeled receptor protein were used for each panel. Nonrecombinant GST, immobilized on glutathione agarose, was used in parallel as a negative control. The radiolabeled receptors remaining bound to the GST or GST-SMRT matrix after washing were eluted, were resolved by denaturing PAGE, and were visualized and quantified by phosphorimager analysis. The amount of radiolabeled receptor bound to the GST or GST-SMRT matrix, relative to the amount of receptor input in each binding reaction, is displayed beneath each panel. Before producing transgenic animals, we validated the characteristics of our PMLRARm4 and RARm4 cDNA constructs, comparing our results with those previously reported. The effects of the m4 mutation on ligand binding had been assessed by evaluating the binding of 3H-tRA to PMLRARm4 or RARm4 in nuclear extracts of transiently transfected Cos-1 cells.31 In these assays, no ligand binding was observed. We used a sensitive protease-resistance assay to determine whether the m4 mutation fully abolished ligand binding. Condensation of a hormone-binding domain of a nuclear receptor around the hormone ligand can result in a protease-resistant core. Gain of protease resistance has been seen with many nuclear hormone receptors and has been used as a measure of ligand occupancy.43,44 Radiolabeled PMLRAR and PMLRARm4 protein were incubated with increasing amounts of trypsin in the absence or presence of 1 µmol tRA. The products were then resolved by denaturing PAGE and visualized by phosphor imaging (Figure 1B). PMLRAR was readily degraded by proteolytic treatment in the absence of hormone, but produced a protease-resistant polypeptide in the presence of hormone. PMLRARm4 also exhibited some protease resistance in the presence of 1 µmol tRA, although it was less resistant than PMLRAR. Ten- to 20-fold higher concentrations of hormone were required to produce a resistant polypeptide from PMLRARm4 than from PMLRAR (data not shown). Although tRA was able to bind weakly to PMLRARm4, when we examined the effects of the m4 mutation on transcriptional activity and association with SMRT corepressor, our results were similar to published analyses. PMLRAR, PMLRARm4, RAR, and RARm4 were transiently expressed in CV-1 cells and transcription from a RARE response element in the absence or presence of tRA was assessed. In contrast to PMLRAR and RAR, the m4 mutant proteins strongly inhibited transcriptional activation (Figure 1C). In cell-free protein assays to measure receptor interaction with SMRT, the m4 mutants, in contrast to RAR, were unable to release the corepressor on addition of 1 µmol tRA (Figure 1D). As has been previously reported,42,45 PMLRAR was itself somewhat less efficient at releasing corepressor than was RAR. The PMLRARm4 and RARm4 cDNAs were cloned into the MRP8 expression vector we had used to generate PMLRAR transgenic mice.32 As with our earlier experiments, transgenic mice were produced in the FVB/N inbred background.38 Injections yielded 7 MRP8-PMLRARm4 and 8 MRP8-RARm4 transgenic animals. Expression of the transgenes The MRP8 promoter element can drive transgene expression in myeloid cells, including myeloblasts, neutrophils, and monocytes.32,36,40 Western blotting of bone marrow was performed using a rabbit polyclonal antiserum raised to human RARF.32 The results are summarized in Tables 1 and 2, and representative data are shown (Figure 2A and B). Although the murine peptide differs from the human by only 4 of 43 amino acids, the antiserum recognizes murine RAR poorly and as a result endogenous murine RAR is not seen on these blots. PMLRARm4 protein was present in 5 of 7 lines of MRP8-PMLRARm4 transgenic mice analyzed. Levels of expression in 2 of the lines appeared comparable to levels of PMLRAR in our highest expressing MRP8-PMLRAR mice (Figure 2A). RARm4 protein was present in 5 of 7 lines of MRP8-RARm4 transgenic mice analyzed. Levels of expression in 2 of the lines appeared to exceed the levels of PMLRAR in our highest expressing MRP8-PMLRAR mice (Figure 2B).                                View this table: [in this window] [in a new window]   Table 1. PMLRARm4 transgenic mice                                View this table: [in this window] [in a new window]   Table 2. RARm4 transgenic mice View larger version (38K): [in this window] [in a new window]   Fig 2. Expression of the transgenes. A, B, Whole cell lysates of bone marrow were subjected to denaturing PAGE and Western blotting using a rabbit polyclonal antihuman RARF domain antibody. Signals corresponding to transgenically expressed PMLRAR and RAR proteins are indicated by arrows. Locations of size markers are indicated by lines. (A) Protein expression in nonleukemic bone marrow of the highest expressing MRP8-PMLRAR transgenic line (Tg556-PR) and in 2 of the MRP8-PMLRARm4 mice, Tg4048-PRm4 (nonleukemic bone marrow) and Tg4099-PRm4 (leukemic bone marrow). 8% SDS-polyacrylamide. (B) Protein expression in nonleukemic bone marrow Tg556-PR and in the marrows of healthy MRP8-RARm4 transgenic mice from 3 lines, Tg4142-Rm4, Tg4151-Rm4, and Tg4192-Rm4. 12% SDS-polyacrylamide. C, Immunofluorescence analysis of bone marrow neutrophilic cells, anti-RARF antiserum and Hoechst 33258, 1300 × . To further substantiate that the transgenes were expressed in myeloid cells, bone marrow was also analyzed by immunofluorescence using the anti-RARF antiserum. Cells with ring-shaped nuclei, as revealed by a fluorescent DNA-binding dye, are primarily neutrophilic. Transgene expression in such neutrophilic cells was observed in both PMLRARm4 and RARm4 transgenic animals (Figure 2C). A range of staining intensity was seen and neutrophilic cells without visibly detectable protein were also present. The speckled nuclear staining present in PMLRARm4 transgenic mice was similar to that seen in PMLRAR transgenic mice. The nuclear staining observed in RARm4 mice lacked the distinct speckles present in the other transgenics. PMLRARm4 transgenic mice develop leukemia Leukemias developed in 4 of 7 founders/lines of MRP8-PMLRARm4 transgenic mice (Table 1), a frequency similar to that encountered previously in MRP8-PMLRAR transgenic mice.32 The latency until leukemia onset, 3 to 11 months, was also comparable to that seen in our MRP8-PMLRAR mice (Table 1; see also Brown et al32). Assessment of leukemia penetrance was hampered by the fact that because of early illness, poor reproduction, or lack of transgene transmission, we did not obtain transgenic offspring for any of the lines in which leukemias developed. Nevertheless, it was apparent that PMLRARm4 could readily initiate leukemia: 3 of 7 independent founder mice developed leukemia and 4 of 5 mice that were reconstituted with the nonleukemic bone marrow of a fourth independent founder also developed leukemia (Table 1). The leukemias arising in the PMLRARm4 transgenic mice were acute leukemias with promyelocytic features. The peripheral blood of the leukemic animals was characterized by anemia, thrombocytopenia, and the presence of leukemic cells at the blast/promyelocyte stage of neutrophilic differentiation (Table 3). The leukemic bone marrows had large numbers of early myeloid cells, many of which had numerous azurophilic primary granules (Figure 3B). These early cells also stained strongly with Sudan Black B (Figure 3D). The morphology and strong Sudan Black B staining were indicative of the promyelocytic character of the leukemic cells. The leukemias caused hepatomegaly and splenomegaly and were invasive, being present not only in the periportal areas of the liver but also invading the liver parenchyma (Figure 3F).                                View this table: [in this window] [in a new window]   Table 3. PMLRARm4 leukemias blood View larger version (146K): [in this window] [in a new window]   Fig 3. Acute leukemia in MRP8-PMLRARm4 transgenic mice. (A, C, E) Samples from control mice. (B, D, F) Samples from leukemic PMLRARm4 mice. (B) Founder 4099. (D) Transplanted leukemia 4048.2. (F) founder 4042. (A, B) Bone marrow, Wright's Giemsa stain, 500 × . (C, D) Bone marrow, Sudan Black B stain, 965 × . (E, F) Liver, Hematoxylin and eosin stain, 200 × . The leukemias were readily transplantable to histocompatible normal mice. Four of the leukemias were each transplanted by intravenous injection into 6 healthy unirradiated nontransgenic FVB/N animals. The cells engrafted and leukemias developed in all 24 recipient mice. The leukemias were subsequently maintained by serial transplantation in vivo and by cryopreservation. The PMLRARm4 leukemias exhibited variability. White blood cell counts ranged from normal to markedly elevated (Table 3). Peripheral blood leukocytes included significant numbers of maturing neutrophilic cells in some but not all cases. Similarly, although the bone marrows of some mice were effaced with promyelocytes, neutrophilic cells maturing beyond the promyelocyte stage were present in other animals (Table 4). Flow cytometric analysis with Gr-1 and Mac-1 markers revealed that leukemia 4099 had the low-level expression pattern typical of leukemias in our PMLRAR transgenic mice, but that the 4042 and 4048.2 leukemias expressed these markers at moderately high levels (Figure 4). The observation that some of the PMLRARm4 leukemias were associated with increased white blood cell counts and significant numbers of maturing neutrophilic cells in the blood and bone marrow contrasts with the leukemic phenotype previously observed in our PMLRAR transgenic mice (Tables 3 and 4).                                View this table: [in this window] [in a new window]   Table 4. PMLRARm4 leukemias bone marrow View larger version (69K): [in this window] [in a new window]   Fig 4. Variable surface marker expression in PMLRARm4 leukemias. Bone marrow cells were stained with Gr-1 and Mac-1 antibodies that recognize myeloid surface antigens. Dead cells were eliminated from the analysis on the basis of staining with propidium iodide. (A) Control. (B) Leukemic mouse 4042 (C) Leukemic mouse 4099 (D) Transplanted leukemia 4048.2. Although PMLRARm4 leukemias were invasive transplantable diseases, they were not always associated with an aggressive clinical course. In our experience with PMLRAR leukemias, the interval between when a leukemic mouse appears ill and when it has progressed to a moribund condition is usually very short, on the order of 1 to 7 days. We were therefore surprised when we noted that mice ill with PMLRARm4 leukemia did not necessarily exhibit rapid deterioration. Leukemic mice 4042 and 4099 were euthanized at the time their leukemias were initially apparent as determined by visible signs of illness. The primary transplant recipients of these 2 leukemias did not rapidly deteriorate after their leukemias became clinically apparent, living with their disease for weeks to months. Leukemic mice 4048.2 and 4104 were not euthanized at the time their leukemias were initially apparent, but were killed more than 2 weeks later. On transplantation, these leukemias were more rapidly fatal (Table 5). Serial transplantation of the leukemias was, in some instances, accompanied by changes in features of the disease that may reflect the accumulation of additional genetic abnormalities. For example, although the first recipients of leukemia 4042 survived with the leukemia for an extended period and exhibited high peripheral white blood cell counts, mice that received the third serial transplant of this leukemia died rapidly without developing a peripheral blood leukocytosis.                                View this table: [in this window] [in a new window]   Table 5. Survival of primary recipients of PMLRAR and PMLRARm4 leukemias All-trans retinoic acid does not cause differentiation of PMLRARm4 leukemias Mice that were recipients of 3 different PMLRARm4 leukemias (4042, 4048.2, 4099) were treated with placebo or tRA to ascertain the effects of the Leu to Pro mutation on tRA responsiveness. For our PMLRAR leukemias, tRA generally causes a rapid rise in the peripheral leukocyte count as leukemic promyelocytes differentiate to mature neutrophils (25-fold average increase in leukocyte count on day 4 of therapy). Morphologic differentiation is readily apparent in the bone marrow of tRA-treated animals (Figure 5A and B; see also Brown et al32) and regression of the leukemia is seen in histologic sections of liver (Figure 5C and D). In contrast, examination of the bone marrow and liver of tRA-treated PMLRARm4 leukemic mice revealed that tRA did not induce morphologic differentiation and disease regression (Figure 5E through L). In addition, unlike PMLRAR leukemias, retinoic acid therapy did not cause a rapid rise in the peripheral white blood cell count (1.5-fold average increase in leukocyte count on day 4 of therapy). View larger version (108K): [in this window] [in a new window]   Fig 5. Retinoic acid response of PMLRARm4 leukemias. Leukemic mice were treated with placebo (A, C, E, G, I, K) or tRA (B, D, F, H, J, L). (A-D) PMLRAR expressing leukemia. (E-H) PMLRARm4 leukemia 4099. (I-J) PMLRARm4 leukemia 4042. (K-L) PMLRARm4 leukemia 4048.2. (A, B, E, F, I-L) Bone marrow, Wright's Giemsa stain, 350 × . (C, D, G, H) Liver, Hematoxylin and eosin stain, 140 × . Effects of 11 days of tRA therapy are shown. We also assessed the clinical effectiveness of tRA in the treatment of leukemias that arose in the PMLRARm4 transgenic mice. To this end, we studied the effect of tRA on survival of mice that were recipients of 2 independent leukemias, leukemia 4048.2 and the aggressive variant of leukemia 4042 that arose on serial passaging. Mice that received leukemic cells by intravenous injection were treated with placebo or tRA when ill. Four recipients of leukemia 4042 treated with placebo died on days 5 and 6 of therapy and 5 mice treated with tRA also died rapidly, on days 5 to 10. Although the difference in survival was statistically significant (P = .04 by log-rank test), the rapid demise of tRA treated animals contrasted with our previous experience with PMLRAR leukemias.32,46 Unexpectedly, although tRA did not cause morphologic differentiation of leukemia 4048.2 (Figure 5K through L), it nevertheless substantially prolonged survival of the leukemic animals: 3 placebo-treated mice died on days 8 and 9 of therapy, whereas 3 tRA-treated mice were still alive at the end of 21 days of tRA treatment (P = .015 by log-rank test). RARm4 transgenic mice are healthy None of the MRP8-RARm4 transgenic mice developed leukemia in up to 18 months of observation (Table 2). This result contrasts with our observations in the MRP8-PMLRAR and MRP8-PMLRARm4 transgenic mice: in these mice, leukemias developed in more than half of the independently derived founders/lines beginning at 3 months of age, and by 10 months of age leukemia had appeared in one third of the mice of the highest expressing PMLRAR line32 and, as noted previously, in 4 of 5 mice derived from a high-expressing PMLRARm4 founder. Although leukemias did not develop in the RARm4 transgenic mice, we investigated whether the RARm4 protein altered neutrophil development in 1 of the highest expressing lines, line 4142. Expression of RARm4 did not alter peripheral white blood cell counts. In the bone marrow, there was a trend toward increased immature neutrophilic cells, but this trend did not reach statistical significance (controls n = 9, line 4142 mice n = 6, P > .05, data not shown). In our previous work, we had observed that MRP8-PMLRAR and MRP8-PEBP2MYH11 transgenic mice exhibited a modest shift in the bone marrow toward immature neutrophilic cells that was accompanied by increased expression of the Mac-1 cell surface antigen (Kogan et al40 and unpublished observations). When stained with Mac-1, the mean fluorescence of the myeloid cells in the bone marrow of RARm4 transgenic mice was 2.4-fold greater than that observed in controls (controls n = 5, line 4142 mice n = 5, data not shown). This increase was statistically significant (P = .002 by Student t test) and is consistent with the trend toward increased immature neutrophilic cells observed by morphologic examination. We also compared the colony-forming units present in the bone marrow of control and RARm4 transgenic mice. Bone marrow cells from groups of 3 healthy untreated mice were grown in methylcellulose cultures in the presence of either G-CSF or a combination of GM-CSF and IL-3. Neither the number of colony-forming units nor the morphology of the cells as assessed on cytospins were significantly different between RARm4 transgenic and control mice (data not shown).     Discussion Top Abstract Introduction Materials and methods Results Discussion References We and others had demonstrated that directing expression of the PMLRAR fusion protein to immature mouse myeloid cells initiated leukemias with promyelocytic features.32,47,48 We have now similarly expressed altered forms of PMLRAR and RAR that are unable to respond to retinoic acid. The results show that retinoic acid responsiveness of PMLRAR, including transcriptional activation by tRA, is dispensable for leukemogenesis. In addition, we found that the ability of the PMLRAR protein to respond to tRA plays an important though perhaps not exclusive role in the therapeutic effects of retinoic acid. Furthermore, our finding that a RAR with dominant negative activity did not readily initiate leukemia suggests that the PML portion of PMLRAR plays an essential role in leukemic transformation. Leukemogenesis cannot be explained by inappropriate transcriptional activation by PMLRAR Alterations in transcription factors have been shown to play a central role in the pathogenesis of leukemias, lymphomas, and other malignancies.49,50 Changes in both transcriptional activation and repression can be important for the pathogenic effects of these alterations. PMLRAR retains the abilities of RAR to repress and to activate transcription of retinoic acid receptor target genes. Although in some settings PMLRAR can act to inappropriately repress transcription, when compared with RAR, it can also increase transcription in the absence or presence of ligand.13,15,17,23-25 The possible role of transcriptional activation in the pathogenesis of APL has not been previously tested. By expressing PMLRARm4 in the myeloid cells of transgenic mice, we directly assessed whether ligand-induced transcriptional activation by PMLRAR is required for leukemic transformation. The ability of PMLRAR variants to initiate leukemia has not been heretofore assessed. Grignani and colleagues45,51,52 examined the ability of altered forms of PMLRAR to inhibit differentiation of the U937 promonocytic cell line. In this cell line, mutations that abolished the ability of PMLRAR to interact with corepressors abrogated the ability of PMLRAR to inhibit differentiation.45 Although studies in U937 cells did not specifically address the role of transcriptional activation in inhibition of differentiation, the PMLRAR variants that were most effective at blocking differentiation retained the ability to act as strong transcriptional activators.52 PMLRARm4 is unable to activate transcription. Our finding that it readily initiates leukemia demonstrates that although PMLRAR can enhance the transcription of RAR target genes, this ability to activate transcription in response to ligand plays no role in leukemogenesis. Retinoic acid responsiveness may influence leukemic phenotype Although RA binding, corepressor release, and transcriptional activation are not required for leukemogenesis, these activities of the PMLRAR protein may influence the characteristics of the leukemias initiated by PMLRAR. Leukemias that developed in our original PMLRAR transgenic mice were characterized by normal white blood cell counts, bone marrow effaced by cells at the promyelocyte stage of maturation, and a rapidly fatal course. In contrast, PMLRARm4 leukemias were more variable: some of these leukemias were characterized by increased white blood cell counts, persistence of neutrophilic maturation beyond the promyelocyte stage, and an indolent clinical course. Because transgenes integrate randomly into the genome, we cannot fully exclude the possibility that the differences between PMLRAR and PMLRARm4 leukemias were due to variation in the level or pattern of protein expression in the independent founders. Furthermore, caution in interpreting such variation is warranted considering the modest number of leukemias that arose in this study. Nevertheless, the differences observed between the PMLRAR and PMLRARm4 leukemias might reflect an effect of endogenous ligands on the behavior of the leukemic cells. Physiologic levels of retinoic acid may bind to PMLRAR and thereby decrease repression or increase activation of target genes. The possibility that ligand-responsiveness may influence leukemic phenotype is supported by a study of leukemias initiated by a PLZFRAR transgene. PLZFRAR is expressed as a result of a t(11;17)(q13;q21) translocation in rare APL patients who have a disease that is clinically resistant to tRA.53,54 Similar to PMLRARm4, PLZFRAR does not activate transcription in response to tRA.25,55 Leukemias in Cathepsin G-PLZFRAR transgenic mice resembled human chronic myeloid leukemia and displayed less of a block in neutrophilic differentiation than leukemias in Cathepsin G-PMLRAR transgenic mice.56 The differences between Cathepsin G-PLZFRAR and Cathepsin G-PMLRAR leukemias are strikingly parallel to those we observed between MRP8-PMLRARm4 and MRP8-PMLRAR leukemias. Given the view that retinoids play a role in fostering neutrophilic maturation, it was unexpected that the fusion proteins that are less responsive to retinoic acid were associated with leukemias with a greater degree of differentiation. Retinoids are not, however, simply differentiative agents. Depending on the experimental system and the maturational state of the cells, retinoids can stimulate or inhibit both proliferation and differentiation of myeloid cells (reviewed in Purton et al57). We speculate that retinoid-responsiveness of PMLRAR facilitates differentiation arrest at the promyelocyte stage of neutrophil maturation. A dominant negative RAR does not appear sufficient to initiate leukemia A number of lines of evidence support the hypothesis that dominant negative inhibition by PMLRAR of RAR may underlie the pathogenesis of APL. First, retinoic acid can enhance neutrophilic differentiation.58,59 Second, dominant negative RAR can inhibit neutrophilic maturation of primary cells.60 Third, comparisons between PMLRAR and PLZFRAR have focused attention on the role of these proteins as transcriptional repressors that can interfere with normal activation of retinoic acid responsive genes.34,42,45,56,61 Fourth, the 4 described translocations involving RAR in APL result in fusions to PML, PLZF, NPM, and NuMA, proteins that do not appear to share common functions. In light of the evidence that transcriptional repression is important in the pathogenesis of APL,34,42,45,56,61 the lack of similarities between the 4 RAR partners raises the possibility that the fusion proteins contribute to APL by acting as dominant negative RARs. The fact that PMLRAR and PMLRARm4 readily initiated leukemias, whereas RARm4 did not, strongly suggests that the PML domain does more than simply confer dominant negative activity onto RAR. The PML domain of PMLRAR may also contribute to transformation by disrupting the normal function of PML, or, alternatively, by altering the DNA binding characteristics of PMLRAR relative to RAR. PML can act as a growth suppressor62-65 and loss of PML predisposes mice to the development of a variety of tumors when the mice are treated with the tumor initiator dimethylbenzanthracene.66 Cells lacking PML grow more quickly than normal cells and are resistant to apoptosis induced by a variety of agents.66,67 These findings indicate that decreased PML function may contribute to transformation by allowing for increased proliferation and increased survival. In support of the hypothesis that disruption of PML activity by PMLRAR contributes to APL is a report that loss of PML increases the penetrance of leukemia in Cathepsin G-PMLRAR transgenic mice.68 An alternative explanation for the differences we have observed in the leukemogenicity of PMLRARm4 and RARm4 is that the PML domain changes binding site specificity69,70: The genes whose transcription is repressed by PMLRARm4 may differ from those repressed by RARm4 and such differences could account for the absence of leukemia in RARm4 transgenic mice. A recent study of retrovirally infected murine bone marrow demonstrated that transcriptional repression by RAR fusion proteins, dominant negative RAR, or unliganded normal RAR can inhibit neutrophilic differentiation and immortalize primary hematopoietic cells.71 These observations support a role for transcriptional repression in APL pathogenesis and raised the possibility that dominant negative RAR would act as potent leukemogen. In vitro immortalization does not, however, always correspond with leukemic behavior in vivo. For example, although HRX-ENL both immortalizes cells and causes leukemia in mice,72 immortalized murine myeloid cell lines such as FDC-P cells are nonleukemic when injected into histocompatible animals.73 The fact that MRP8-PMLRARm4 but not MRP8-RARm4 mice developed leukemia is not inconsistent with the hypothesis that transcriptional repression is necessary for leukemic transformation. Furthermore, although our results show that the leukemogenic potential of PMLRAR extends beyond its ability to inhibit RAR function, our findings cannot be interpreted to indicate that dominant negative RAR would not contribute to leukemia when expressed in mice under conditions other than those we have used. The expression of RARm4 in the bone marrow cells of our transgenic mice had a modest impact on neutrophil differentiation. The lack of a strong effect of the MRP8-RARm4 transgene compared with prior in vitro studies of retrovirally expressed dominant negative RAR could reflect a number differences in experimental methods, including protein expression levels in hematopoietic progenitors, in vitro growth conditions, and mouse strain. In addition, expression of the transgene was heterogeneous and its effects on differentiation of total bone marrow may consequently have been masked by the presence of neutrophilic cells with low or absent RARm4 protein. Our results are, however, concordant with studies of mice that lack RAR1 and RAR (which comprise the vast majority of RARs expressed in myeloid cells): RAR1/, RAR/ mice displayed no apparent hematopoietic defect in vivo and the neutrophilic cells from these mice showed a modest maturation defect in vitro.74 Retinoic acid binding to PMLRAR is essential for retinoic acid-induced differentiation of leukemic promyelocytes The leukemias that arose in the PMLRARm4 transgenic mice did not differentiate in response to retinoic acid. The m4 mutation was originally described in a subclone of the NB4 cell line selected to grow in retinoic acid. Our results parallel the previously observed association of the m4 mutation with resistance to differentiation, and further demonstrate that the ability of tRA to cause differentiation of leukemic cells requires direct effects of tRA on the PMLRAR fusion protein. Interestingly, tRA may have been of some therapeutic benefit despite the fact that differentiation of the leukemic blasts was not observed. Such a therapeutic effect of tRA has been observed in at least 1 case of human APL exhibiting marked in vitro resistance to the differentiative effects of tRA.28 The mechanism by which tRA was of benefit in these individual murine and human leukemias is not known. However, the possibility that tRA might improve survival even when it does not cause morphologic differentiation draws attention to the fact that novel therapies may have unanticipated effects that contribute to their clinical impact. This finding also suggests that tRA therapy may, in combination with other agents, continue to be of some benefit to APL patients identified as having resistant disease.     Acknowledgments We thank Daphne Haas-Kogan and H. Jeffrey Lawrence for critical reading of the manuscript, and Meijuan Zhou for technical assistance.     Footnotes Submitted June 25, 1999; accepted October 28, 1999. Supported by grants CA 4338 and CA 75985 from the National Institutes of Health and by funds from the G.W. Hooper Research Foundation. S.C.K. is a recipient of a Burroughs Wellcome Fund Career Award. Reprints: Scott C. Kogan, Department of Laboratory Medicine, Room M524, Box 0100, 505 Parnassus Ave, University of California, San Francisco, CA 94143-0100. The publication costs of this article were defrayed in part by page charge payment. 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