Id1 and Id2 Are Retinoic Acid Responsive Genes and Induce a G0/G1 Arrest in Acute Promyelocytic Leukemia Cells.

Abstract

Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which overcomes the differentiation arrest and induces terminal granulocytic differentiation of the leukemic blasts. In 98% of the cases of APL, the leukemic cells express a promyelocytic leukemia (PML)- retinoic acid receptor a (RARa) fusion protein as a result of a t(15;17) chromosome translocation. Previously, we have identified Id1 and Id2 as direct retinoic acid target genes being upregulated after ATRA stimulation in the APL cell line NB4 as well as in primary leukemic cells from APL patients. Id (inhibitor of DNA-binding) proteins act as antagonists of basic helix-loop-helix (bHLH) transcription factors by trapping them in heterodimeric complexes, thereby inhibiting DNA-binding and gene transactivation. Various bHLH proteins are pivotal in the control of differentiation and proliferation in various tissues (like muscle and nerve). We have studied the expression pattern of E2A, an ubiquitously expressed bHLH protein, which is generally considered as a promiscuous heterodimerization partner of other, more tissue restricted bHLH proteins. The expression of E2A was high in untreated APL cells and strongly downregulated upon ATRA stimulation. The simultaneous upregulation of Id1 and Id2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells upon treatment with ATRA. To assess the importance of Id1 and Id2 induction for the neutrophilic differentiation of the cells, we have overexpressed both proteins in the APL cell line NB4 using amphotropic retroviral transduction. Ectopic expression of Id1 and Id2 resulted in respectively 27% (n=3, SD= 9%) and 48% (n=3, SD=25%) inhibition of clonogenic growth in semi-solid medium, compared to vector-transduced control cells. Apart from the reduction in the number of colonies, overexpression of Id1 and Id2 did not alter the ATRA sensitivity of APL cells. NB4 liquid cultures revealed that Id1 and Id2 overexpression resulted in inhibition of proliferation and an increase of the percentage of cells in G0/G1, without having an effect on differentiation or apoptosis. These results indicate that Id1 and Id2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.