ABSTRACT
To elucidate the possible mechanism of differentiation and/or apoptosis in NB4, K562 cells induced by tributyrin (TB), a histone deacetylase inhibitor (HDACi), the level of acetylated histone H3 was detected by Western blot, p21(WAF1) expression was detected by semi-quantitative RT-PCR. The results showed that histone H3 hyperacetylation was detected in NB4 (or K562) cells after treatment with TB 0.1 mmol/L (or TB 0.5 mmol/L) for 16 hours in a dose-dependent manner. p21(WAF1) dose-dependently increased at RNA level in these two cell lines treated by TB 0.1 mmol/L. The level of p21(WAF1) mRNA increased at 2 hours after TB treatment, reaching peak at 16 hours, and maintaining for 48 hours. In conclusion, the mechanism of differentiation and apoptosis in NB4, K562 cells induced by tributyrin may associate with its up-regulation of acetylated histone and p21(WAF1) mRNA level.
Subject(s)
Humans , Acetylation , Apoptosis , Blotting, Western , Cell Differentiation , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Dose-Response Relationship, Drug , Enzyme Inhibitors , Pharmacology , Gene Expression Regulation, Neoplastic , Histone Deacetylase Inhibitors , Histone Deacetylases , Metabolism , Histones , Metabolism , K562 Cells , Leukemia , Genetics , Metabolism , Pathology , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Triglycerides , PharmacologyABSTRACT
To establish leukemic cell lines stably transfected by RbAp46 gene, electroporation was performed after optimizing the transfection condition for suspended cells. Under conditions of low voltage and high capacitance, RbAp46 was transfected into U937 by electroporation. Individual clones selected with G418 for 3 weeks were isolated. The integration and the protein levels of the exogenous RbAp46 in transfectants were determined by PCR and Western blot analysis, respectively. The subclone expressing high level of RbAp46 was then established. Viability of transfected cells was assayed by trypan blue exclusion. Cell number was counted daily to determine the growth rate. The results showed that growth rate of U937 cell lines expressing exogenous RbAp46 was about 50% lower than that in control. It is concluded that leukemic cell lines stably expressing exogenous RbAp46 were established and overexpression of RbAp46 inhibits the growth of U937 leukemic cells.
Subject(s)
Humans , Blotting, Western , Carrier Proteins , Genetics , Cell Proliferation , Electroporation , Nuclear Proteins , Genetics , Retinoblastoma-Binding Protein 7 , Transfection , U937 Cells , WT1 Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of tributyrin (TB), a histone deacetylase inhibitor, on the growth, differentiation and apoptosis of SHI-1 leukemia cells and explore its possible mechanism.</p><p><b>METHOD</b>Cell proliferation and viability were determined by cell counting, trypan blue dye exclusion. Cell morphological analysis, Annexin binding, DNA electrophoresis, expression of CD11b and CD14, NBT reduction were performed to evaluate differentiation and apoptosis of SHI-1 cells. The level of acetylated histone H3 was detected by Western blot and p21(WAF1/CIP1) expression by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>TB inhibited the proliferation and viability of SHI-1 cells in a time-dose dependent manner. The morphology of SHI-1 cells cultured in the presence of 0.1 mmol/L TB for 72 hs was more mature with higher NBT positivity and up-regulated expressions of CD11b and CD14 than that of control group. Exposed to 0.5 - 1.0 mmol/L TB for 48 hs, SHI-1 cells exhibited the morphological hallmarks of apoptosis, the increasing of Annexin binding and the DNA ladder on gel electrophoresis. The level of acetylated histone H3 and p21(WAF1) mRNA extracted from SHI-1 cells were increased by the treatment of TB.</p><p><b>CONCLUSION</b>TB can inhibit proliferation, induce differentiation and apoptosis of SHI-1 cells. The mechanism may associate with its up-regulation of acetylated histone and the expression of p21(WAF1).</p>
Subject(s)
Humans , Acetylation , Apoptosis , Blotting, Western , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Enzyme Inhibitors , Pharmacology , Gene Expression , Histone Deacetylase Inhibitors , Histone Deacetylases , Metabolism , Histones , Metabolism , Leukemia, Monocytic, Acute , Genetics , Metabolism , Pathology , Reverse Transcriptase Polymerase Chain Reaction , Triglycerides , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To identify a mutation G2113-->A in the glycoprotein (GP)IX gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis.</p><p><b>METHODS</b>Allele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-IXG2113A harboring the mutation G2113-->A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPIbalpha, GPIbeta and GPIX or mutant GPIX, respectively. Expression of GPIbalpha and GPIX in transfected CHO cells were analysed with flow cytometer. GPIbalpha and GPIX in the cytoplasma of transfected CHO cells were analysed by immunostaining and Western blotting.</p><p><b>RESULTS</b>The patient was found to be homozygosity of the substitution, her mother and her brother be heterozygous. Expressions of GPIbalpha and GPIX in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma.</p><p><b>CONCLUSION</b>The mutation of Ala139(GCC)-->Thr(ACC) in the GPIX did not affect synthesis and assembly of GPIb/IX complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.</p>
Subject(s)
Adult , Animals , Cricetinae , Female , Humans , Bernard-Soulier Syndrome , Genetics , Blotting, Western , CHO Cells , Mutation , Platelet Glycoprotein GPIb-IX Complex , GeneticsABSTRACT
WT1 gene encodes a zinc finger transcription factor that regulates transcription of its downstream genes. Some of target genes for WT1 are involved in regulating both cell cycle and cellular proliferation and differentiation. However, WT1 itself is regulated by its upstream genes such as NF-kappaB and GATA-1. Thus there exists a pathway of transcriptional regulation mediated by WT1, which controls development of hematopoietic system. Leukemia results from disrupting the homeostasis among hematopoietic proliferation, differentiation and apoptosis, which is often the consequence of an inappropriate expression of transcription factors and subsequent disruption of the normal gene expression pattern. This article reviews the relationship between the WT1-mediated pathway of transcriptional regulation and leukemia.