Study On The Alteration Of Nuclear Matrix Proteins During Induced Differentiation Of Human Hepatocarcinoma SMMC-7721 Cells

In this study,5mmol/L hexamethylene biacetamide(HMBA ) was used to induce the differentiation of human hepatocarcinoma SMMC-7721 cells,and its effects were examined.On this base,by use of the methods of subcellular proteomics, immunocytochemistry and molecular biology,we studied systematically the changes of nuclear matrix proteins during differentiation of SMMC-7721 cells induced by HMBA,analysed their co-localizational relationship with products of related oncogenes and tumor suppressor genes,and speculated their functions to the induced differentiation of SMMC-7721 cells.It's useful for us to find out the role of differential expressed nuclear matrix proteins on induced differentiation of tumor cell and explore the molecular mechanisms of carcinogenesis and malignant phenotypic reversion in a relatively systematical level.The results revealed that the proliferation of SMMC-7721 cells were inhibited after 7 days of treatment by HMBA.The reduplication time ratio of treated cells to untreated cells was 1.8.The percentage of cells in G₀/G₁ phase increased from 40.6% to 70.6%while decreased from 27.4%to 6.9%in S phase.The cell cycle of SMMC-7721 cells were arrested in G₀/G₁ obviously.Light and electric microscope observation had showed that after treated with 5 mmol/L HMBA,the ratio of nuclear to cytoplasm lessened,the volume of cell increased,the number of nucleoli decreased. The number of microvilli and the heterochromatin decreased while euchromatin and free ribosomes increased.The endoplasmic reticulum,mitochondfion and golgi body all developed.It was revealed by immunocytochemistry assay that the expression level of oncogenes including c-myc,c-fos,c-erbB-2,bcl-2,mtp53 were downregnlated,while the expression level of tumor suppressor genes such as Rb,p21 and p27 were upregulated.By cellular selective extaction and whole-mount SEM/TEM observation,we found that after HMBA inducement,the NM-IF filaments of SMMC-7721 cells were concentrated and distributed uniformly.The heterogeneous population of filaments,including highly branched utrathin filaments could also be seen in the regular meshwork.The connection between the two kinds of filaments and the relatively thin,condensed and sharply demarcated lamina composed of intermediate-sized filaments was relatively fastened.Meanwhile,9 NM proteins changed remarkably during SMMC-7721 cell differentiation.Four proteins,i.e. mutant pystl,hypothetical protein,nucleophosmin,and LBP were downregulated, whereas four other proteins,eIF6,p44 subunit,β-tubulin 2C,and sin3b were upregulated with the last one,SFRS1 only found in the differentiated SMMC-7721 cells.Two proteins-nucleophosmin and prohibitin had been found existed and localized in the nuclear matrix,as well as its altered expression,by use of western blot, immunofluorescence staining and immune electron microscopy analysis.It also showed that nucleophosmin and prohibitin were colocalized with oncogene c-fos and c-myc products,as well as tumor suppressor gene mtp53 and Rb products.Both of their expression level and localization changed during the differentiation of SMMC-7721 cells.It is concluded that the human hepatocarcinoma SMMC-7721 cells could be induced into differentiation by treatment with HMBA,the malignant morphological and ultrastructural characteristics were reversed,the configuration and composition of nuclear matrix was altered.Some nuclear matrix proteins which were importantant to gene expression and regulation or cell signal transduction have changed in their expression level and subcellular localization during the differentiation.The colocalization of nucleophosmin and prohibitin with products of related oncogenes such as c-myc,c-los and tumor suppressor genes such as Rb,p53 suggested the pathway and mechanisms in which specific nuclear matrix proteins regulated the proliferation and differentiation of the human hepatocarcinoma SMMC-7721 cells. These results indicated that HMBA could interfere the activities of some oncogenes and tumor suppressor genes,regulate the expression of important nuclear matrix proteins,then cause cell cycle arrest,and induce human hepatocarcinoma cells into differentiation.Our study provides proofs and a new way to explore the mechanisms of induced differentiation of tumor cells and cell carcinogenesis and its reversion. Besides,this study provides several potential target proteins for cancer diagnosis and cancer therapy.