Differential gene regulation of WT1 protein tumor antigen and the components of the antigen processing machinery by interferon-gamma signaling in myeloid leukemia

Abstract

2587 Background: Expression of high levels of Wilms Tumor Protein-1 (WT1) is associated with poor prognosis in myeloid leukemias and myelodysplastic syndromes.We studied the effects of interferon-γ (Inf-γ) on the myeloid cell line K562 which express the WT1 mRNA and the lymphoid cell line LG2 which does not express the transcripts. The biological effects of Inf-γ are mediated through activating the Janus kinase (JAK)-STAT pathway, which in turn, can activate a large number of genes by binding to the interferon-stimulated response element (ISRE). Methods: The mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) using published primer sequences and quantitated by a real time PCR (RQ-PCR). The WT1 protein expression was detected by western blotting with a commercially available WT1 monoclonal antibody. Intracellular flow cytometry with monoclonal antibodies was used to study the components of the antigen processing machinery (APM) as established before. Results: WT1 is down-regulated in K562 cells by treating K562 cells with Inf-γ. In a timed experiment WT1 mRNA is not seen after 36 hours of treatment both by RT-PCR and RQ-PCR. Western blotting for the protein done at 48 hours confirms the absence of the protein. No apoptosis is seen in these cells as shown by BrDU staining. Intracellular flow cytometry for the APM show- up regulation of the interferon responsive class I heavy chain and Tapasin stabilizing the heavy chain with the beta-2 microglobulin. WT1 Down-stream proteins such as bcl-2 and c-myc are also downregulated. There is no change in the expression of the chaperone proteins such as calreticulin, calnexin and HSP70. However the heat shock proteins HSP90 and HSP27 are downregulated. Similar effects on the WT1 are not seen in the control cell line LG2 or after treatment of the K562 cells with PMA, TNF-alpha or 5-azacytidine. Conclusions: The downregulation of WT1 is Inf-γ specific suggesting involvement of the STAT pathways. WT1 acts a differentiation signal for leukemia cells, which has implications in targeted treatment of leukemia. Preliminary results from patient sample study confirm some of these findings. No significant financial relationships to disclose.