Abstract
The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.
Highlights
The nucleotide sequence reported in this paper has been submitted to the EMBL/GenBankTM/EBI Data Bank with accession number AJ272324
These data strongly suggest that increased PRAM-1 mRNA expression correlates with the capacity of leukemia cells to undergo granulocytic differentiation when treated with all-trans retinoic acid (ATRA)
Upon finding that SLP-76 was expressed constitutively and that SKAP-HOM was induced by ATRA in NB4 cells (Fig. 4B), we investigated whether PRAM-1 associates with these proteins
Summary
Cell Lines, Culture, and Differentiation—NB4 [20], NB4.306 [23], and U937-PR9 and U937-MT cell lines [17] were cultured in RPMI 1640 medium with 10% fetal bovine serum (Life Technologies, Inc.) and 2 mM L-glutamine (Life Technologies, Inc.). Expression of the PRAM-1 and control -actin mRNA in differentiating cells was analyzed by semi-quantitative PCR using a GeneAmp PCR System 9600 (PerkinElmer Life Sciences) with the following primers corresponding to distinct exon sequences: sense (5Ј-CCTCAGTTCAGCAAGCCGCCAGGAG-3Ј) and antisense (5Ј-CCAGGGGGAGTGGTTGGTTTTCCAG-3Ј) for PRAM-1; sense (5Ј-CCTCGCCTTTGCCGATCC-3Ј) and antisense (5Ј-GGATCTTCATGAGGTAGTCAGTC-3Ј) for -actin. A polyclonal sheep antiserum (Elmira Biologicals) was raised against the carboxyl-terminal 134 amino acids of human PRAM-1 fused with glutathione S-transferase (generated by cloning the corresponding PRAM-1 coding sequence into the pGEX vector (Amersham Pharmacia Biotech)). Immunoprecipitation—After one-step preclearing (30 min, 4 °C) with GammaBind G-Sepharose (Amersham Pharmacia Biotech), antibodies (1 l of antiserum or 1 l of ascites fluid) were added to the cell protein extract, in a binding buffer adjusted to 20 mM Tris-HCl, pH 7.5, 0.25 M NaCl, and 0.1% Nonidet P-40. Enzymatic activity was detected using the chemiluminescence reagent plus kit (PerkinElmer Life Sciences) and autoradiography
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