Proteomic profiling drug-induced apoptosis in non-small cell lung carcinoma: identification of RS/DJ-1 and RhoGDIalpha.

The growing knowledge of the tight connection between apoptosis and cancer has lead to an explosion of research revolving around apoptotic induction with chemotherapeutic agents and small molecule inhibitors. The chemotherapeutic agent paclitaxel (Taxol) activates mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase and, combined with MEK inhibition, synergistically enhances apoptosis. Here we implement a proteomic approach using two-dimensional gels coupled with mass spectrometry to identify proteins altered with this coordinated combination treatment. We found that the combined treatment of paclitaxel and MEK inhibitor uniquely altered the proteins RS/DJ-1 (RNA-binding regulatory subunit/DJ-1 PARK7) and RhoGDIalpha (Rho GDP-dissociation inhibitor alpha). Functional proteomic analysis by exogenous expression or short interfering RNA targeting confirmed a role in survival and apoptosis for these proteins. Analysis of primary lung tumors with matched adjacent normal tissue confirmed RS/DJ-1 overexpression in non-small cell lung carcinoma. This study shows the power of proteomic profiling coupled with functional analysis for the discovery of novel molecular targets and potential cancer cell-specific biomarkers.

Differential control of apoptosis by DJ-1 in prostate benign and cancer cells.

DJ-1 is a conserved protein reported to be involved in diverse cellular processes ranging from cellular transformation, control of protein-RNA interaction, oxidative stress response to control of male infertility, among several others. Mutations in the human gene have been shown to be associated with an autosomal recessive, early onset Parkinson's disease (PARK7). The present study examines the control of DJ-1 expression in prostatic benign hyperplasia (BPH-1) and cancer (PC-3) cell lines in which DJ-1 abundance differs significantly. We show that while BPH-1 cells exhibit low basal level of DJ-1 expression, stress-inducing agents such as H(2)O(2) and mitomycin C markedly increase the intracellular level of the polypeptide. In contrast, DJ-1 expression is relatively high in PC-3 cells, and incubation with the same cytotoxic drugs does not modulate further the level of the polypeptide. In correlation with the expression of DJ-1, both cytotoxic agents activate the apoptotic pathway in the prostatic benign cells but not in PC-3 cells, which are resistant to their action. We further demonstrate that incubation of BPH-1 cells with TNF-related-apoptosis-inducing-ligand/Apo-2L (TRAIL) also enhances DJ-1 expression and that TRAIL and H(2)O(2) act additively to stimulate DJ-1 accumulation but synergistically in the activation of the apoptotic pathway. Time-course analysis of DJ-1 stimulation shows that while DJ-1 level increases without significant lag in TRAIL-treated cells, there is a delay in H(2)O(2)-treated cells, and that the increase in DJ-1 abundance precedes the activation of apoptosis. Unexpectedly, over-expression of DJ-1 de-sensitizes BPH-1 cells to the action of apoptotic-inducing agents. However, RNA-interference-mediated silencing of DJ-1 expression results in sensitization of PC-3 cells to TRAIL action. These results are consistent with a model in which DJ-1 is involved in the control of cell death in prostate cell lines. DJ-1 appears to play a differential role between cells expressing a low but inducible level of DJ-1 (e.g., BPH-1 cells) and those expressing a high but constitutive level of the polypeptide (e.g., PC-3 cells).

The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease.

Mutations in DJ-1, a human gene with homologues in organisms from all kingdoms of life, have been shown to be associated with autosomal recessive, early onset Parkinson's disease (PARK7). We report here the three-dimensional structure of the DJ-1 protein, determined at a resolution of 1.1 A by x-ray crystallography. The chain fold of DJ-1 resembles those of a bacterial protein, PfpI, that has been annotated as a cysteine protease, and of a domain of a bacterial catalase whose role in the activity of that enzyme is uncertain. In contrast to PfpI, a hexameric protein whose oligomeric structure is essential for its putative proteolytic activity, DJ-1 is a dimer with completely different intersubunit contacts. The proposed catalytic triad of PfpI is absent from the corresponding region of the structure of DJ-1, and biochemical assays fail to detect any protease activity for purified DJ-1. A highly conserved cysteine residue, which is catalytically essential in homologues of DJ-1, shows an extreme sensitivity to radiation damage and may be subject to other forms of oxidative modification as well. The structure suggests that the loss of function caused by the Parkinson's-associated mutation L166P in DJ-1 is due to destabilization of the dimer interface. Taken together, the crystal structure of human DJ-1 plus other observations suggest the possible involvement of this protein in the cellular oxidative stress response and a general etiology of neurodegenerative diseases.