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

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 MGcS0-3 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-TOFMS） 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 MGcS0-3 cells compared to control. Eleven of which were identified. Seven proteinsactin, prohibitin, porin 31HL, heterogeneous nuclear dbonucleoprotein 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.

THE DIFFERENTIATION OF HUMAN GASTRIC ADENOCAR-CINOMA CELL LINE MGc80-3 INDUCED BY DIBUTYRYL cAMP IN VITRO

For providing some experimental basis in establishing malignant phenotypic reversed indexes of gastric carcinoma cells, human gastric adenocar-cinoma cell line MGc80-3 was induced by dBcAMP in vitro to appraise the effect of gastric carcinoma cell differentiation by chemical inducers.Under light microscope, MGc80-3 cells, after treated with 1 mM dBcAMP, tended to be flat and disperse, and their volume gradually enlarged, with their uncleus relatively smaller and their shape rather regular. Morphological changes, like norma differentiated epithelial cells, were observed. The cells attached firmly, grew slowly, their growth curve showed inhibitory rate amounted to 52.87%, and cellular division exponent displayed their peak value 1.5 times less than that of MGc80-3 cells. It was clear that dBcAMP could effectively inhibit the multiplication activity of MGc80-3 cells. After dBcAMP treatment, remarkable changes of cell surface charges was indicated by cell electrophoresis, the ratio dropped to 3.043 from 3.988, and their re-tardant ratio reached up to 31.2%. cAMP content in cells after this treatment, detected by cAMP and cGMP radioimmunoassay, was enhanced by 2.42 times, and cAMP/cGMP ratio, by 1.73 times. Thus, cAMP level within MGc80-3 cells was raised obviously by dBcAMP. Heterotransplantation experiments showed that tuntorigenic rate of MGc80-5 cells (transplanted subcutaneously to BALB/c mice) amounted to 100%, and that of the cells after this treatment was only 5.6%. Their tumorigenic ability was extremely reduced.These results confirmed that dBcAMP was able to change malignant phenotypic characteristics of MGc80-3 cells and produce a reversed alteration: Thus, it has a remarkable inductive effect in differentiating gastric carcinoma cells. All these characteristics were also considered as the reference indexes in appraising reversed effect for the homologous cancer cells.

CHEMOSENSITIVITY OF MGc80-3 HUMAN GASTRIC ADENOCARCINOMA CELLS AND ITS CLINICAL SIGNIFICANCE

In the present study, the chemosensitivity of MGc80-3 human gastric adenocarcinoma cells was determined by means of colony-forming assay and the in vitro activities of 10 anticancer drugs were examined on the basis of the clinically achievable peak plasma drug concentration. The results showed that MGc80-3 cells were most sensitive to mitomyc'n C, adriamycin and 5-fluorouracil, being consistent with the response noted in clinical gastric cancer. This cell line may retain its original drug sensitivity and may be useful in screening for new compounds with activity against this disease.

微管-微丝系统的改变对培养的MGc 80-3人胃癌细胞粘液分泌的影响

本工作用PAS反应显示粘液蛋白的方法对细胞内微管微丝变化时人胃腺癌MGc 80-3细胞粘液分泌活动进行了研究。秋水仙酰胺、低温(2~4℃)及细胞松弛素B(CB)处理后,细胞内微管微丝解聚,粘液分泌活动受到抑制,当细胞从低温回置37℃温育,微管重新组装后,粘液分泌又得到恢复。Taxol短时间处理对MGc 80-3细胞粘液分泌有促进作用、但长时间处理后,由于细胞内微管的高度浓集化,从而破坏了细胞内微管网络结构(CMTC),使粘液分泌活动停止,说明人胃腺癌MGc80-3细胞的粘液分泌活动依赖于细胞内CMTC和微丝的存在。毛果芸香碱能直接刺激培养的MGc 80-3细胞的粘液分泌,提示人胃腺癌MGc 80-3细胞膜也可能存在类胆碱能受体。硫杂脯氨酸对人胃腺癌MGc 80-3细胞的增殖具有抑制作用,但对其粘液分泌具有强烈的刺激作用,这种刺激作用可通过加速粘液的排放而实现。毛果芸香碱和硫杂脯氨酸对MGc 80-3细胞粘液分泌的刺激作用也依赖于细胞内微管微丝的存在,一旦微管微丝解聚,其刺激粘液分泌的作用便消失。db-cAMP对MGc 80-3细胞的粘液分泌也有促进作用。CB单独处理或CB与秋水仙酰胺同时处理,都能抑制MGc 80-3细胞的粘液分泌,但CB单独处理后,分泌颗粒多聚集于细胞边缘,而CB与秋水仙酰胺同时处理后,分泌颗粒则散在分布于胞质中,提示在MGc 80-3细胞内微丝对粘液的排放,而微管对分泌颗粒的输送起主要作用。