Identification of DJ-1/Park-7 as a Determinant of Tnfα-Induced and Stroma-Dependent Apoptosis in Leukemia Using Mass Spectometry and Phosphopetide Analysis.

Abstract

The bone marrow microenvironment provides essential signals for the fate of normal hematopoietic and for leukemic cells. Contact with marrow stroma, which is part of the microenvironment, is generally thought to convey anti-apoptotic signals to (clonal) leukemia cells. Patients with low-grade myelodysplastic syndrome (MDS) early in the disease course show high rates of apoptosis in normal and clonal marrow cells, mediated by tumor necrosis factor alpha (TNFα) and other cytokines. As MDS advances and evolves to leukemia, clonal cells tend to become apoptosis resistant. We showed previously that the leukemia-derived cell line KG1a was resistant to TNFα-mediated apoptosis, but TNFα did induce caspase-3 activation and apoptosis in KG1a cells when co-cultured with the human marrow stroma cell line HS5 (derived from healthy marrow). Apoptosis was contact dependent and required expression of TNF receptor 1 on KG1a cells. Identical results were obtained in co-cultures with primary stroma cells. Gene expression profiling of KG1a cells showed that stroma contact resulted in significant upregulation of genes involved in apoptosis, including PYCARD and p53. To further dissect the relevant signaling pathways, we used a PhosphoScan proteomic LC-MS (Liquid chromatography-mass spectrometry) method to identify proteins that were phosphorylated in response to stroma contact. In parallel to KG1a we examined the parent cell line KG1, which is sensitive to TNFα mediated apoptosis. We determined the phosphorylation sites in proteins within the leukemic cell lines using MS2 and MS3 scans. Database searches were performed with X! Tandem and Mascot and results analyzed by PeptideProphet using data from a synthetic doubly-phosphorylated peptide as control. In KG1a cells cultured without stroma support, the peptide DJ-1/Park-7 was highly phosphorylated, and expression of p53 was inhibited as indicated by decreased levels of p53 mRNA and protein. In co-culture with stroma, KG1a cells expressed higher levels of p53 protein, and levels of phosphorylated DJ-1/ Park-7 were undetectable over a time course of 30 min to 24 hours. In apoptosis-sensitive KG1 cells constitutive DJ-1/Park-7 phosphorylation (in the absence of stroma contact) was undetectable, and p53 was expressed at higher levels than in KG1a cells, consistent with the observed activation of caspase-3 and induction of apoptosis in KG1 cells. Taken together, these data suggest that phosphorylation of DJ-1/Park-7, originally identified as an oncogene product involved in cellular transformation, oxidative stress responses, and transcriptional regulation, was associated with repression of p53 and resistance to TNFα-mediated apoptosis. The relevance of DJ-1/Park-7 (and other genes identified by the PhosphoScan proteomic method) in primary MDS cells is currently being investigated at the molecular and functional levels.