Analyses of CREB and ICER expression in acute childhood leukemias.
Authors: Martina Pigazzi, Emanuela Ricotti, Eva Trevisson, Cristina Baldoin, Giuseppe Germano, Leonardo Salviati, Maurizio Aricò, Giuseppe Basso
Journal: Cancer Research (2006)

Proc Amer Assoc Cancer Res, Volume 47, 2006

5654 

Cyclic adenosine monophosphate response-element binding protein (CREB) is a transcription factor that regulates gene expression controlling cell differentiation, proliferation and survival. A role for CREB in growth factor dependent survival has been shown in glucose homeostasis, learning and memory processes. Recently, it has been demonstrated that is implicated in myeloid cell transformation. We used Real Time PCR, Western blot and ELISA to study CREB expression in 231 bone marrow samples obtained from acute lymphoid (ALL) and acute myeloid (AML) childhood leukemia at diagnosis and during therapy, compared to nonleukemia and healthy samples. We studied CREB role in leukemia analyzing its phosphorylation state and its transcriptional activity by EMSA. We induced the expression of its endogenous antagonist ICER (inducible cAMP early repressor) and performed quantitative gene expression analysis by TaqMan Gene Expression Assays. Results revealed that CREB protein is significantly upregulated in 84% of ALL and in 66% of AML at diagnosis (p<0.001). Moreover CREB is in the active phosphorylated form at diagnosis of acute leukemias and it was always transcriptionally active bound to its consensus DNA cAMP response element (CRE) exclusively at diagnosis of leukemias and not constitutively. Nevertheless, CREB was strongly downregulated in leukemic specimens in documented remission during chemotherapy and spiked up in bone marrow going to relapse. Moreover, it was downregulated also in non hematological disorders together with the healthy controls. We hypothesize that ICER , the endogenous antagonist of CREB, deserves a special consideration in direct activation of gene expression. ICER intact DNA-binding domain binds specifically to CREs functioning as a potent repressor of CREB activity. We tested whether ICER expression is related to CREB over expression in leukaemia. ICER was down-regulated in leukemia at diagnosis, but not in remission, nor in controls. In order to evaluate whether ICER disrupts CRE-mediated transcription, we tested CyclinA1 expression, which is modulated positively by CREB and repressed by ICER. We constructed an expression vector and induced ICER protein in Hela cell line. Results revealed that when ICER was induced, CREB protein disappeared. Moreover, gene expression analyses revealed that after 48 hours of induction, a series of target genes expression was downregulated. We thus hypothesized that CREB over expression might up-regulate target genes, affecting cell proliferation and survival at diagnosis of leukaemia. Insufficient ICER expression might fail to counteract these events. These findings represent an important first step in the understanding of the physiological processes linked to the cAMP/CREB/ICER pathway. The future understanding of ICER role in blocking cAMP activation pathway or CREB target genes expression will elucidate this pathway and may help understanding leukemogenesis.