ABSTRACT
To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.
Subject(s)
Humans , Apoptosis , Blotting, Western , Cell Cycle , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21 , Enzyme Inhibitors , Pharmacology , Flow Cytometry , Histone Deacetylase Inhibitors , Hydroxamic Acids , Pharmacology , Leukemia, Myeloid , Metabolism , PathologyABSTRACT
Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.
Subject(s)
Animals , Humans , Rabbits , Antibodies , Allergy and Immunology , Cloning, Molecular , Escherichia coli , Genetics , HeLa Cells , Immune Sera , Allergy and Immunology , Recombinant Fusion Proteins , Allergy and Immunology , Telomeric Repeat Binding Protein 1 , Genetics , Allergy and ImmunologyABSTRACT
Glycophorin A (GPA) is one of the important molecular markers in studies of somatic cell mutations. To investigate the relationship between the gamma-irradiation and the frequency of GPA variation, the frequency of variant erythrocytes at the GPA locus was determined in peripheral blood of 3 subjects with accidental whole-body gamma-irradiation. The biological dose of individuals was 2.5, 2.9 and 1.9 Gy estimated by the chromosome aberration assay, respectively, and the frequency of GPA variation was 3.9, 4.3 and 4.1 times greater than that from normal controls, respectively. Our results suggest that the variant frequency of erythrocyte GPA was increased. On account of the GPA gene mutations are preserved in hematopoietic stem cells during all irradiated individual's life, the frequency of GPA variation could be used as a permanent marker for mutagenesis of radiation.