Altered expression of zinc finger proteins, ADAMs, and integrin‐related proteins following treatment of cultured human cells with a low concentration of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine

Abstract

Proteomic analysis is an important approach to characterize the proteome and study protein functions. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be overlooked by conventional biochemical techniques. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is an alkylating agent that can induce nontargeted mutagenesis in treated cells, although the mechanism remains unclear. Using an efficient proteomic method, we identified several cellular proteins that are responsive to low-concentration MNNG treatment in human FL cells. After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images then were analyzed with 2D analysis software. More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). Thirty-one proteins only detected in MNNG-treated or control cells were subjected to in-gel digestion with trypsin and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using peptide mass fingerprinting. Eighteen of theses proteins have been identified, including several zinc finger proteins, two members of the ADAMs (a disintegrin and metalloprotease domain) family, and two integrins. Most of these proteins have unknown functions and their involvement in the cellular responses to alkylating agents have not been reported. Therefore, our findings may offer new insights into the mechanisms of low-concentration MNNG-induced nontargeted mutagenesis and these proteins may serve as new biomarkers for detecting exposure of human populations to environmental carcinogens.

Proteomic analysis of cellular responses to low concentration N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in human amnion FL cells

“…Ratio changes for the up-and downregulated spots are listed in Table I. For the index number and an example of a gel image, see Figure 1 in Jin et al [2003]. The magnitude of ratio changes ranged from 0.2 (downregulation; spot 70) to 5.7 (upregulation; spot 1246).…”

“…Following silver staining, the gels were digitized prior to computer-based matching and quantitative analysis with image analysis software. As described in our previous publication [Jin et al, 2003], a total of 1,145 Ϯ 58 protein spots were detected in control FL cells, and 1,214 Ϯ 44 spots in MNNG-treated FL cells within a pI range of 4 -7 and a size range of ϳ10 -100 kDa. The intensity of 32 protein spots was altered after MNNG treatment.…”

“…The procedures were essentially the same as described by Jin et al [2003]. Briefly, samples were prepared from lysed cells, and the proteins were run on a 2-DE system, following the manufacturer's instructions (Amersham Biosciences, Uppsala, Sweden).…”

“…Proteomic analysis com-bining two-dimensional gel electrophoresis (2-DE) and mass spectrometry provides a high-throughput method for screening proteins responsive to a specific stimulus and is probably the most appropriate approach for such a study. We previously reported that the expression of over 60 proteins was altered in MNNG-treated FL cells; among them, 31 proteins were only detected in MNNG-treated or control cells, and 18 of these were identified by matrixassisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using peptide mass fingerprinting [Jin et al, 2003]. In this study, the other 32 proteins that were up-or downregulated by MNNG treatment were subjected to further analysis, and among these, 18 proteins have been identified by peptide mass fingerprinting.…”

Sphingolipids are involved in N-methyl-N′-nitro-N-nitrosoguanidine-induced epidermal growth factor receptor clustering

“…NMethyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, is a potent carcinogen and is able to induce various cellular responses, including the activation of intracellular signal transduction pathways and changes in protein expression profile [22][23][24]. One unique response induced by MNNG treatment is the clustering of TNFR and EGFR in both African green monkey kidney Vero cells and human amnion FL cells [24,25].…”