Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

Abstract

Aim: To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy.

Methods: Nuclear matrix proteins were selectively extracted from MGc80-3 cells treated with or without hexamethylamine bisacetamide (HMBA), and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis and submitted for database searching using Mascot tool.

Results: The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGc80-3 cells compared to control. Eleven of which were identified. Seven proteins--actin, prohibitin, porin 31HL, heterogeneous nuclear ribonucleoprotein A2/B1, vimentin, ATP synthase, and heat shock protein 60 were downregulated, whereas three proteins--heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated, and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells.

Conclusion: The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

Figure 1

2-D PAGE gels of nuclear matrix proteins from MGc80-3 cells, Coomassie blue-stained A: MGc80-3 cells; B: MGc80-3 cells exposed to 5 mmol/L HMBA for 7 d. Arrows pointed to changed proteins. Disappeared protein: C33; downregulated proteins: C19, C20, C23, C27, C28, C29, C30, C38, C39, C40, C41, C70; new protein: S78; upregulated proteins: S4, S44, S53, S54, S72, S74, S75.

Figure 2

Enlarged maps of changed nuclear matrix proteins from MGc80-3 cells, Coomassie blue-stained A–H: nuclear matrix from control cells; I-P: nuclear matrix from HMBA-treated cells. Arrows pointed to changed proteins. C33 disappeared, while C19, C20, C23, C27, C28, C29, C30, C38, C39, C40, C41, C70 were downregulated in the HMBA-treated cells; S78 was a new protein, whereas S4, S44, S53, S54, S72, S74, S75 were upregulated after HMBA treatment.

Figure 3

Relative expression level of changed nuclear matrix proteins. A: Relative expression level of disappeared or downregulated proteins in the nuclear matrix fractions of MGc80-3 cells treated with HMBA; B: Relative expression level of new or upregulated proteins in the nuclear matrix fractions of MGc80-3 cells treated with HMBA. C33 was disappeared, while C19, C20, C23, C27, C28, C29, C30, C38, C39, C40, C41, C70 were downregulated in the HMBA-treated cells; S78 was a new protein, whereas S4, S44, S53, S54, S72, S74, S75 were upregulated after HMBA treatment. Relative expression levels were shown using Melanie software. Values are mean±SE for two experiments.