Kun Sang Chang

PML, a growth suppressor disrupted in acute promyelocytic leukemia

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PMLs transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

Regulation of AML2/CBFA3 in Hematopoietic Cells through the Retinoic Acid Receptor α-Dependent Signaling Pathway

AML2 is a member of the acute myelogenous leukemia, AML family of transcription factors. The biologic functions of AML1 and AML3 have been well characterized; however, the functional role of AML2 remains unknown. In this study, we found that AML2 protein expressed predominantly in cells of hematopoietic origin is a nuclear serine phosphoprotein associated with the nuclear matrix, and its expression is not cell cycle-related. In HL-60 cells AML2 expression can be induced by all three natural retinoids, all-trans-retinoic acid (RA), 13-cis-RA, and 9-cis-RA in a dose-dependent manner. A synthetic retinoic acid derivative, 4HPR, which neither activates RA receptor (RAR) α nor retinoic X receptor α was unable to induce the expression of AML2. A RAR-selective activator, TTNPB, induced AML2 expression similar to RA. Our study further showed that AGN193109, a potent RARα antagonist, suppressed AML2 expression induced by RA and that a retinoic X receptor pan agonist AGN194204 had no effect on its expression. Taken together, these studies conclusively demonstrated that the expression of AML2 in HL-60 cells is regulated through the RARα-specific signaling pathway. Our study further showed that after all-trans-retinoic acid priming, AML2 expression could be augmented by vitamin D3. Based on these studies we hypothesize that AML2 expression is normally regulated by retinoid/vitamin D nuclear receptors mainly through the RARα-dependent signaling pathway and that it may play a role in hematopoietic cell differentiation.

Comparison of outcome in acute myelogenous leukemia patients with translocation(8 ; 21) found by standard cytogenetic analysis and patients with AML1/ETO fusion transcript found only by PCR testing

Patients with normal-karyotype acute myelogenous leukemia (NKAML) may have undetected genetic abnormalities that could affect prognosis. Screening for known AML-specific genetic abnormalities using the reverse transcription polymerase chain reaction (RT-PCR) may help in arriving at a more definitive prognosis. To test this hypothesis, 104 patients without translocation (8;21) and inversion(16), as shown by standard cytogenetic (SC) analysis, were screened for these two genetic abnormalities using RT-PCR. Western blot analysis for the AML1/ETO fusion protein and fluorescent in situ hybridization (FISH) analysis for t(8;21) were performed in patients for whom we had samples. The characteristics and outcome after high-dose cytarabine containing treatments in five patients with t(8;21) shown by RT-PCR alone were then compared to 21 patients with t(8;21) detected using SC analysis. Eight of the 104 patients had masked t(8;21) and none had masked inv(16), as shown by RT-PCR. Five of 54 patients with NKAML had a detectable AML1/ETO fusion RNA transcript. Western blot analysis showed the AML1/ETO fusion protein in four of the seven patients for whom we had samples among the eight with masked t(8;21) shown by RT-PCR. All patients with t(8;21) shown by RT-PCR had negative FISH results. Ninety percent (n = 19) of the patients with t(8;21) shown by SC analysis and 40% (n = 2) of the patients with t(8;21) shown by RT-PCR alone achieved a complete remission (Pvalue 0.03). These data suggest that the outcome of NKAML patients with t(8;21) shown by RT-PCR is not equivalent to patients with t(8;21) by SC studies.

Characterization of the ETO and AML1–ETO proteins involved in the 8;21 translocation in acute myelogenous leukemia

Abstract: The AML1 and ETO genes are disrupted by the nonrandom chromosomal translocation t(8;21) in acute myelogenous leukemia (AML). While the AML1 gene encodes a transcription factor indispensable for definitive hematopoiesis, the biological function of ETO is unknown. To understand the role of ETO and AML1–ETO in the pathogenesis of AML, the full length cDNAs of ETO and AML1–ETO were cloned and antibodies against AML1 and ETO proteins have been developed in our laboratory. Western blot analysis showed that ETO and AML1–ETO were identified as 70 kDa and 94 kDa proteins, respectively, and that both proteins, like AML1, were associated with the nuclear matrix. To examine whether the t(8;21)‐positive AMLs expressed a 94‐kDa AML1–ETO, protein fractions isolated from leukemia blasts of 10 patients with t(8;21)‐positive AML and the Kasumi‐1 cells were analyzed by Western blotting. The 94 kDa AML1–ETO fusion protein was detected in all samples. However, this fusion protein was not detectable in all 40 patients with t(8;21)‐negative AMLs. The biological significance of AML1–ETO was examined in K562 cells, which stably overexpress AML1–ETO. We found that AML1–ETO blocked the erythroid differentiation of K562 cells induced by low doses of Ara‐C. Thus, t(8;21)‐positive AMLs appear to overexpress the AML1–ETO fusion protein, which may be responsible for differentiation block and leukemogenesis in AML.

Promyelocytic leukemia protein PML inhibits Nur77-mediated transcription through specific functional interactions

The promyelocytic leukemia protein PML is a tumor and growth suppressor and plays an important role in a multiple pathways of apoptosis and regulation of cell cycle progression. Our previous studies and others also documented a role of PML in transcriptional regulation through its association with transcription coactivator CBP and transcription corepressor HDAC. Here, we showed that PML is a potent transcriptional repressor of Nur77, an orphan receptor and a member of the steroid receptor superfamily of proteins. We found that PML represses Nur77-mediated transactivation through a physical and functional interaction between the two proteins. PML interacts with Nur-77 in vitro in a GST-pull down assay and in vivo by coimmunoprecipitation assay. PML/Nur77 colocalized in vivo in a double immunofluorescent staining and confocal microscopic analysis. Our study further showed that the coiled–coil domain of PML interacts with the DNA-binding domain of Nur77 (amino acids 267–332). Electrophoretic mobility shift assay demonstrated that PML interferes with Nur77 DNA binding in a dose-dependent manner. This study indicates that PML interacts with the DNA-binding domain of Nur77 and represses transcription by preventing it from binding to the target promoter. This study supports a role of PML/Nur77 interaction in regulating cell growth and apoptosis.

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.

The promyelocytic leukemia protein represses A20-mediated transcription.

The promyelocytic leukemia (PML) protein is a tumor suppressor that is disrupted by the chromosomal translocation t(15;17), a consistent cytogenetic feature of acute promyelocytic leukemia. A role of PML in multiple pathways of apoptosis was conclusively demonstrated using PML−/− animal and cell culture models. In a previous study, we found that PML sensitizes tumor necrosis factor-induced apoptosis in tumor necrosis factor (TNF)-resistant U2OS cells. This finding helped to explain the mechanism of PML-induced apoptosis. The zinc finger protein A20 is a target gene of NFκB inducible by TNFα, and it is a potent inhibitor of TNF-induced apoptosis. In the this study, we demonstrated that PML is a transcriptional repressor of the A20 promoter and that PML represses A20 expression induced by TNFα. We showed that PML inhibits A20 transactivation through the NFκB site by interfering with its binding to the promoter. We also showed that stable overexpression of A20 inhibits apoptosis and caspase activation induced by PML/TNFα. The results of this study suggest that A20 is a downstream target of PML-induced apoptosis and supports a role of A20 in modulating cell death induced by PML/TNFα in TNF-resistant cells.