Alteration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation.
OBJECTIVE
To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells.


METHODS
Cells cultured with or without 5 x 10(-3) mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www.matrixscience.com).


RESULTS
The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched ultrathin 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, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pyst1, hypothetical protein, nucleophosmin 1, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, beta-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells.


CONCLUSIONS
The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament (NM-IF) system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.
Tang
Jian
J
The Key Laboratory of Chinese Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian Province, China.
Niu
Jing-Wen
JW
Xu
Dong-Hui
DH
Li
Zhi-Xing
ZX
Li
Qi-Fu
QF
Chen
Jin-An
JA
D000081:Acetamides / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000503:physiopathology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D054642:Dual Specificity Phosphatase 6; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007382:Intermediate Filaments / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000503:physiopathology*; D015530:Nuclear Matrix / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology; D000090243:Nucleophosmin; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000235:genetics / Q000502:physiology
D000081:Acetamides; D000970:Antineoplastic Agents; C109284:EIF6 protein, human; D039642:Eukaryotic Initiation Factors; C000716967:NPM1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D014404:Tubulin; D000090243:Nucleophosmin; C517035:DUSP6 protein, human; D054642:Dual Specificity Phosphatase 6; D017027:Protein Tyrosine Phosphatases; C014026:hexamethylene bisacetamide