Study On The Changes Of Nuclear Matrix Proteins During The Differentiation Of The Human Neuroblastoma SK-N-SH Cells Induced By Retinoic Acid

In this study,1μmol/L Retinoic Acid(RA) was used to induce the terminal differentiation of human neuroblastoma SK-N-SH cells,and its effects were investigated.On this base,by use of the methods of subcellular proteomics, immunocytochemistry and cellular molecular biology,we studied systematically the changes of nuclear matrix proteins during differentiation of SK-N-SH cells induced by RA,analysed their co-localizational relationship with products of related oncogenes and tumor suppressor genes,and speculated their functions to the induced differentiation of SK-N-SH 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 experiment results showed that after treaed with 1μmol/L RA,the proliferation rate of SK-N-SH cells was inhibited,the inhibition rate amounts to 57.93%,and the cell cycle was arrested in G₀/G₁ phase;the morphology and ultrastructure of SK-N-SH cells underwent a significant change and appeared as normal differentiated neuronal cells:cells became multipolar with several neurites,cell bodies were smaller, with long axonal processes extending from one cell to another to form ganglia;The number of microvilli and the heterochromatin decreased while euchromatin and free ribosomes increased.The endoplasmic reticulum,mitochondrion and Golgi body all developed.The immunocytochemistry assey also revealed that the expression level of oncogenes including c-myc,c-los were downregulated,and the expression level of tumor suppressor genes including p53,p27 were upregulated,and at the same time, the expression of the neuronal markers of terminal differentiation,including NSE, SYP and MAP2 increased greatly after treatment.By cellular selective extaction and whole-mount SEM/TEM observation,we found that after treatment with RA,the NM-IF filaments of SK-N-SH 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,52 nuclear matrix proteins(NMPs),41 of which were identified changed significantly during SK-N-SH differentiation;The changes of Nucleophosmin,Prohibitin,Vimentin and hnRNP A2/B 1 differentially expressed nuclear matrix protein during differentiation induced by RA,was further confirmed by western-blot and had been found existed and localized in the nuclear matrix by use of immunofluorescence staining and immune electron microscopy analysis.The 4 specific NMPs were co-localized with oncogenes c-myc and c-fos products,as well as tumor suppressor genes p53 and Rb products. The expression level and location of them were changed following the proliferation and differentiation of SK-N-SH.The co-localization of neuronal markers with oncogenes and tumor suppressor genes also changed obviously during the differentiation.Our study showed that the human neuroblastoma SK-N-SH cells were induced into terminal differentiation after treatment with RA,as the proliferation of SK-N-SH cells were inhibited,the cell cycle were arrested in G₀/G-1,the malignant morphological and ultrastructural characteristics were reversed,the configuration of nuclear matrix-intermediate filament was altered,the expression of the neuronal phenotype markers were highly increased.Some nuclear matrix proteins which play an important role in DNA replication and repair,cell cycle regulation,gene expression and regulation,have changed in expression level and location during the differentiation. The co-localization of nucleophosmin,prohibitin vimentin and hnRNP A2/B1 with products of neuroblastoma associated oncogenes c-myc,c-fos and tumor suppressor genes p53,Rb suggested the pathway and mechanisms in which specific nuclear matrix proteins regulated the proliferation and differentiation of the human neuroblastoma SK-N-SH cells.In conclusion,1μmol/L RA can induce SK-N-SH cells into terminal differentiation by regulating the activities of some oncogenes and antioncongenes,altering the expression of some key nuclear matrix proteins,arresting cell cycle,and facilitating the expression of neuronal phenotype markers.Our findings will help to elucidate the signaling pathways and mechanisms of neuroblastoma cell growth and differentiation as well as carcinogenesis,and provides a new way to explore the mechanisms of induced differentiation of tumor cells and cell carcinogenesis and its reversion.Further study of the proteins identified here will be useful for the development of clinical therapies targeting neuroblastoma.