The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties.

The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RARalpha). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RARalpha sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RARalpha. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid-inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RARalpha in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RARalpha. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RARalpha. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RARalpha to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype. (Blood. 2000;95:2683-2690)

Differentiation of t(5;17) variant acute promyelocytic leukemic blasts by all-trans retinoic acid.

All-trans retinoic acid (ATRA) induces differentiation of acute promyelocytic leukemic (APL) blasts from patients with t(15;17) APL. However, blasts from patients with the t(11;17) variant do not differentiate in response to ATRA. Our group has identified a variant of APL characterized by t(5;17) and expression of the NPM-RAR fusion gene product. From case reports it has been difficult to establish whether ATRA induces clinical responses in patients with this variant. In order to determine whether t(5;17) blasts differentiate with ATRA, we harvested mononuclear bone marrow cells from a patient with t(5;17) APL at time of relapse and cultured them in medium containing ATRA. Morphologic analysis of cytospins after 7 days of culture revealed that 60% of cells in the ATRA-treated culture had differentiated into mature neutrophilic forms, as opposed to less than 1% in the control culture. Seventy-three percent of cells acquired NBT positivity after exposure to ATRA, compared with 1% in the control culture. These results indicate that t(5;17) blasts retain the ability to terminally differentiate in response to retinoic acid.

The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion.

We have studied an acute promyelocytic leukemia (APL) patient with a variant t(5;17)(q32;q12). This translocation fuses the gene for the nucleolar phosphoprotein nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). Two alternatively spliced transcripts are expressed, which differ in 129 bases immediately upstream of the RARA sequence. The NPM sequences contained in the shorter NPM-RAR cDNA are identical to the NPM sequences contained in the NPM-ALK fusion gene expressed in t(2;5) lymphomas. The RARA sequences are the same as the RARA sequences found in the PML-RAR and PLZF-RAR fusion seen in t(15;17) and t(11;17) APL, respectively. Both NPM-RAR transcripts fuse NPM and RARA sequence in the same reading frame, to generate translation products of 57 kD and 62 kD. Both NPM-RAR proteins are expressed in the patient's leukemic cells, along with wild-type RARA derived from the uninvolved allele. In transcriptional assays using a retinoic acid response element reporter construct, both NPM-RAR fusion proteins act as retinoic acid-dependent transcriptional activators. This case defines a third class of APL rearrangements, all of which generate fusion proteins of RARA.

Reduction of NMDA receptors with dithiothreitol increases [3H]-MK-801 binding and NMDA-induced Ca2+ fluxes.

1. We have investigated the modulation of N-methyl-D-aspartate (NMDA) receptor activation by the sulphydryl redox reagents dithiothreitol (DTT) and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). 2. Increases in [3H]-MK-801 binding produced by glutamate, glycine and spermidine were enhanced by DTT (2mM) and diminished by DTNB (0.5 mM). 3. The inhibition of [3H]-MK-801 binding by CGS 19755 and 7-chlorokynurenate was not altered by 2 mM DTT. However, the potency of the competitive polyamine antagonist, arcaine, was decreased by DTT. 4. NMDA-induced Ca2+ fluxes into primary cultures of rat forebrain neurones were enhanced by DTT in a DTNB-reversible fashion. In addition to augmenting the magnitude of NMDA-induced increase in intracellular free Ca2+, 10 mM DTT also prolonged the duration of the Ca2+ signal. However, DTT had no effect on the increase in Ca2+ produced by depolarizing neurones with 50 mM KCl. 5. These studies show that the reduction of disulphide bonds on the NMDA receptor complex by DTT increases activation. The precise site of these groups remains unclear but they are unlikely to form an integral part of the glutamate, glycine or polyamine binding domains. The enhancement of the activation of the NMDA receptor by DTT is associated with increased Ca2+ fluxes. The possible pathophysiological consequences of receptor reduction are discussed.

Role of lipid solubility in the interaction of drugs with the N-methyl-D-aspartate receptor.

We have examined the hypothesis that phenothiazines and tricyclic antidepressants interact with the N-methyl-D-aspartate (NMDA) receptor by mimicking the actions of Zn2+. Using [3H]MK801 binding to well-washed rat brain membranes, we found a disparity between the concentrations of drug required to inhibit [3H]MK801 binding and those necessary to alter dissociation of [3H]MK801 from its binding site. These data suggest that the Zn2+ site is probably not the principle site by which tricyclic drugs inhibit NMDA receptor responses. To determine whether the effects of tricyclic drugs on the dissociation of [3H]MK801 could be explained simply by membrane stabilization, we examined the effect of a series of alcohols. Ethanol, isopropanol, butanol, hexanol, and heptanol all inhibited [3H]MK801 binding. However, with the exception of heptanol, none of the molecules altered the dissociation rate. Thus, while lipid solubility may be an important factor underlying the interactions of some drugs with the NMDA receptor, it is not sufficient to explain the effects of tricyclic antidepressants and phenothiazines in this system.

Depression in family practice patients.

Multiple factors have been described as significant contributors to depression in medical patients. This study attempts to assess the relative significance and interrelationship of variables associated with depression. A group of 199 family practice patients were studied. Using a multivariate research design, significant depressive symptoms were found in 41% of the sample. A stepwise multiple regression analysis revealed that the five most important factors associated with depression, in order of significance, were socioeconomic status, recent stress, use of birth control pills, serious physical illness, and distant life events. This study supports the thesis that depression is a final common pathway syndrome in which biologic and social forces coalesce into syndrome expression. The primary care practitioner needs to be aware of the multiple risk factors for depression to develop effective detection and intervention strategies.