Exploring (novel) gene expression during retinoid-induced maturation and cell death of acute promyelocytic leukemia.

During recent years, reports have shown that biological responses of acute promyelocytic leukemia (APL) cells to retinoids are more complex than initially envisioned. PML-RARalpha chimeric protein disturbs various biological processes such as cell proliferation, differentiation, and apoptosis. The distinct biological programs that regulate these processes stem from specific transcriptional activation of distinct (but overlapping) sets of genes. These programs are sometimes mutually exclusive and depend on whether the signals are delivered by RAR or RXR agonists. Furthermore, evidence that retinoid nuclear signaling by retinoid, on its own, is not enough to trigger these cellular responses is rapidly accumulating. Indeed, work with NB4 cells show that the fate of APL cells treated by retinoid depends on complex signaling cross-talk. Elucidation of the sequence of events and cascades of transcriptional regulation necessary for APL cell maturation will be an additional tool with which to further improve therapy by retinoids. In this task, the classical techniques used to analyze gene expression have proved time consuming, and their yield has been limited. Global analyses of the APL cell transcriptome are needed. We review the technical approaches currently available (differential display, complementary DNA microarrays), to identify novel genes involved in the determination of cell fate.

Genomic organization of the JEM-1 (BLZF1) gene on human chromosome 1q24: molecular cloning and analysis of its promoter region.

The Jem-1 (JEM-1, HGMW-approved symbol BLZF1) gene mapping to human chromosome 1q24 codes for a ubiquitously expressed 3-kb mRNA, translated in a 45-kDa nuclear protein. Recent studies have shown a deficient expression of this gene in acute promyelocytic leukemia (APL). However, treatment with retinoids was able to upregulate JEM-1 mRNA in maturing NB4 leukemia cells. Here, we report the characterization of the structural organization of JEM-1. By hybridization screening of a human genomic library derived from blood mononuclear cells, five overlapping genomic DNA clones were isolated. These clones extend over 34 kb of the human genome and comprise the complete JEM-1 gene and a 4-kb 5'flanking region. Determination of the exon-intron structure of Jem-1 revealed seven exons whose junctions with introns exhibited typical splice sequences. A shorter transcript (Jem-1s, 1.3 kb) generated by exon 3 extension and polyadenylation was identified. Its translation generated a 23-kDa protein that exhibited a cytoplasmic localization. 5'RACE-PCR identified a major transcription start site (TSS) located at 403 nt upstream of the ATG. Computer analysis of the 1. 8-kb 5'flanking region showed that it lacks a TATA box, Inr motifs or DPE motifs, but it contains a typical CCAAT box located 95 bp upstream of the TSS. Sequencing also revealed potential cis-acting elements for multiple transcription regulators including Sp1, GATA, C/EBP, AP-1, and Pu1. No retinoic acid receptor elements or retinoic X receptor elements were detected. This 1.8-kb DNA sequence showed a strong constitutive promoter activity determined by a luciferase-reporter gene assay in transiently transfected HeLa cells. Retinoids further increased luciferase expression 2.7-fold. We demonstrated that the 1-kb distal sequence contains yet unidentified elements reducing constitutive transcription. Thus, the maximal constitutive promoter activity was assigned to a -432 + 101 region overlapping the TSS. These data support the idea of a constitutive expression of JEM-1, but a negative regulation in APL released by retinoids.

Gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells.

To elucidate the molecular mechanism of all-trans-retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) cells, the gene expression patterns in the APL cell line NB(4) before and after ATRA treatment were analyzed using complementary DNA array, suppression-subtractive hybridization, and differential-display-polymerase chain reaction. A total of 169 genes, including 8 novel ones, were modulated by ATRA. The ATRA-induced gene expression profiles were in high accord with the differentiation and proliferation status of the NB(4) cells. The time courses of their modulation were interesting. Among the 100 up-regulated genes, the induction of expression occurred most frequently 12-48 hours after ATRA treatment, while 59 of 69 down-regulated genes found their expression suppressed within 8 hours. The transcriptional regulation of 8 induced and 24 repressed genes was not blocked by cycloheximide, which suggests that these genes may be direct targets of the ATRA signaling pathway. A balanced functional network seemed to emerge, and it formed the foundation of decreased cellular proliferation, maintenance of cell viability, increased protein modulation, and promotion of granulocytic maturation. Several cytosolic signaling pathways, including JAKs/STAT and MAPK, may also be implicated in the symphony of differentiation. (Blood. 2000;96:1496-1504)

JEM-1, a novel gene encoding a leucine-zipper nuclear factor upregulated during retinoid-induced maturation of NB4 promyelocytic leukaemia.

Retinoid-induced proliferation causing hyperleukocytosis is a severe complication of retinoid therapy in t(15;17) acute promyelocytic leukaemia. The molecular basis of this phenomenon is unknown. It is possible that the transiently enhanced cell proliferation results from RA-induction of growth regulatory genes. Using Differential Display of cDNAs from NB4 cells we have identified Jem, a novel gene transcript which is upregulated by retinoids during the early proliferative response in maturating cells but not in resistant cells. A 2.7 kb cDNA was cloned and sequenced. The open reading frame contains a 400 amino acid sequence corresponding to a novel 45 kDa basic protein (pI 8.9). The JEM DNA sequence is detected by FISH on human chromosome 1 at q24. The Jem peptide sequence shows a 'leucine-zipper' dimerisation motif with limited homology to Fos/Jun and ATF/CREB proteins and several putative phosphorylation sites. An atypical basic region may correspond to an unknown DNA-binding domain. The C-terminal end of Jem spans a long stretch featuring a PEST motif. After transfection into COS cells, the Jem protein shows a ponctuated nuclear localisation. We hypothesise that this novel nuclear factor may act as a transcription factor, or a coregulator, involved in either cell growth control and/or maturation.