Up-regulation of Acid Sphingomyelinase during Retinoic Acid-induced Myeloid Differentiation of NB4, a Human Acute Promyelocytic Leukemia Cell Line

Abstract

All-trans-retinoic acid (ATRA) induces myeloid differentiation of a human promyelocytic leukemia cell line, NB4, but does not affect its subclone NB4/RA harboring a point-mutated ligand-binding domain (AF2) in retinoic acid receptor ␣ (RAR␣) gene. We found that ATRA induced the 4-fold elevation of acid sphingomyelinase (ASMase) activity 24 h after treatment in NB4 cells, but not in NB4/RA cells. ATRA did not affect neutral sphingomyelinase activity in either NB4 or NB4/RA. Upon treatment with ATRA, ceramide, the product of an ASMase reaction, accumulated in NB4 cells. Northern blot analysis showed a marked elevation of the ASMase mRNA 8 h after ATRA treatment, reaching a plateau at 24 h. Regulation of ASMase gene expression was studied by a promoter analysis using luciferase reporter assay. The 5-upstream flanking region of human ASMase gene (؊519/؉300) conjugated with the luciferase gene was introduced into COS-7 cells. Luciferase activity in transformed cells markedly increased in response to ATRA stimulation when the wild type RAR␣ or the PML/RAR␣ hybrid protein was co-expressed. Deletion experiments revealed that a short sequence at the 5-end (؊519/؊485) was indispensable for the ATRA response. Within this short region, two retinoic acid-responsive element-like motifs (TGCCCG and TCTCCT) and one AP2-like motif (CCCTTCCC) were identified. Deletion and base-substitution experiments showed that all three motifs are required for the full expression induced by ATRA. Electrophoresis mobility shift assays with the nuclear extract of ATRA-treated NB4 cells showed that proteins were bound specifically to the probe being mediated by all three motifs in the promoter sequence.

Bioactive Sphingolipids in Response to Chemotherapy: A Scope on Leukemias

“…Immunologic clearance of leukemic cells by TRAIL-induced apoptosis was observed after pulsing dendritic cells with -galactosylceramide but not prior to pulsing, suggesting that other sphingolipids might have roles for other effective therapeutic approaches [190]. Interestingly, ceramides were shown to be potent to induce differentiation in AML cells which somewhat show the characteristics of immature stem cells [131,191]. Some gangliosides, neolacto-series and GM3 in particular, have similar roles for the differentiation of leukemic cells [192,193].…”

Retinoic acid inhibits the infectivity and growth of Chlamydia pneumoniae in epithelial and endothelial cells through different receptors

“…2, panels C and D). Interestingly, ATRA has also been reported to increase the intracellular ceramide concentration, by increasing the production of acid sphingomyelinase, which results in hydrolysis of sphingomyelin to ceramide [21]. It is known that Chlamydia utilize host cell derived sphinoglipids for synthesis of the inclusion membrane [22].…”

Arsenic trioxide inhibits ATRA-induced prostaglandin E2 and cyclooxygenase-1 in NB4 cells, a model of acute promyelocytic leukemia

“…21 ATRA-induced COX-1 upregulation may be mediated by the Sp1 transcription factor, which is significantly upregulated during myeloid differentiation, and has been reported to mediate ATRA responsiveness of several genes devoid of RAR-responsive element (RARE) motifs. [27][28][29] In that sense, the COX-1 promoter contains two Sp1-binding sequences but no canonical RARE. 30 Previous reports described a prodifferentiating role of PGE 2 , the main product of COX-1 activity, in myelopoiesis.…”