The Overexpression of ANKHD1 in Acute Leukemia Cells and Its Association with SIVA-1, a Proapoptotic Protein.

Ankyrin-repeat-containing proteins regulate multiple cellular functions including transcriptional and cell-cycle regulation, ion channel, cell survival and cell signaling and participate in protein-protein interactions via their repeat motifs. Ankyrin Repeat and KH Domain Containing 1-ANKHD1, was first identified in LNCaP, a human prostate cancer cell line. However, protein expression patterns or protein interactions of ANKHD1 have not yet been described. The aim of this studied was to characterize the mRNA and protein expression patterns of ANKHD1 in normal hematopoietic cells and acute leukemia cells, and also to identify new proteins that interact with ANKHD1 in hematopoietic cells. The National Ethical Committee Board approved the study and informed-written consent was obtained from all subjects. Real Time RT-PCR was performed and increased levels of ANKHD1 mRNA were detected in acute leukemia cell lines; KG-1, HEL, K562, NB4, HL-60 Jurkat, MOLT4, Raji, Daudi and Namalwa (up to 10-fold increase), compared to normal bone marrow cells. Additionally, ANKHD1 mRNA expression was significantly higher in samples from patients with a diagnosis of acute myeloid leukemia (n = 30) (medians: 1.97 versus 1.05, P = 0.004) when compared to normal hematopoietic cells (n = 9), and in samples from patients with a diagnosis of acute lymphoblastic leukemia (n = 7) (medians: 2.53 versus 1.05, P = 0.005) when compared to normal hematopoietic cells (n = 9). Regarding protein expression, immunoblot analysis detected a very low expression of ANKHD1 in Peripheral Blood Mononuclear Cells (PBMC) of normal donors. Conversely, a high expression of the protein was detected in the human acute leukemia cell lines, KG-1, HEL, K562, NB4, HL-60 Jurkat, MOLT4, Raji, Daudi and Namalwa. Laser confocal analysis of K562 and Jurkat cell lines and PBMC showed that ANKHD1 is located in the cytoplasm of leukemia and normal hematopoietic cells. This cytoplasmatic localization of ANKHD1 was further confirmed by immunoblotting performed with anti-ANKHD1 antibody and tissue fractions of the Jurkat cell line. In an attempt to identify the proteins associated with ANKHD1 in hematopoietic cells, yeast two-hybrid screening was used and AH109 was transformed with PGBKT7-ANKHD1 (amino acids 1130–1243) and used to screen a Matchmaker pACT2-cDNA library prepared from normal human bone marrow (Clontech). The screening identified a new protein interaction between ANKHD1 and SIVA-1. This interaction was further confirmed by immunoprecipitation and Western Blotting analysis in the Jurkat cell line. The finding that ANKHD1 interacts with SIVA-1, which has been recently identified as a proapoptotic protein up regulated in leukemia cells, as well as the high expression of ANKHD1 in acute leukemia, described in our study, gives rise to the hypothesis that ANKHD1 protein may be involved in leukemogenesis. The identification of new disease-specific targets for acute myeloid leukemia immunotherapy expands treatment options and increases our chances of successfully treating this heterogeneous disease and lowering the unacceptably high mortality rate.