ABSTRACT
OBJECTIVE: To investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence. METHODS: Chromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-beta-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA. RESULTS: Mitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-beta-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells. CONCLUSION: Mitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.
Subject(s)
Aminoglycosides/pharmacology , Antibiotics, Antineoplastic/pharmacology , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/pathology , Enediynes/pharmacology , Liver Neoplasms/enzymology , Liver Neoplasms/pathology , Telomerase/metabolism , Apoptosis/drug effects , Azure Stains , Benzimidazoles , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cellular Senescence/drug effects , Chromatin/metabolism , DNA, Neoplasm/analysis , Dose-Response Relationship, Drug , Genome, Human/genetics , Humans , Membrane Potential, Mitochondrial/drug effects , Mitosis/drug effects , Phenotype , Propidium , Time Factors , beta-Galactosidase/metabolismABSTRACT
AIM: Mitotic cell death has been focused on in tumor therapy. However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells. METHODS: Growth curve was established by MTT assay. Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle. Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively. RESULTS: Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-beta-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells. Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication. CONCLUSION: Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.
Subject(s)
Aminoglycosides/pharmacology , Antibiotics, Antineoplastic/pharmacology , Carcinoma, Hepatocellular , Cell Death/drug effects , Centrosome/drug effects , Liver Neoplasms , Cell Line, Tumor , Cell Nucleus/drug effects , Cellular Senescence/drug effects , Chromatin/drug effects , Enediynes , Humans , Mitochondria , Mitosis/drug effects , Phenotype , Polyploidy , Spindle Apparatus/drug effectsABSTRACT
OBJECTIVE: To analyze the effects of aging or advanced glycation on gene expression in the cerebrum and spleen of female C57BL/6J mice. METHODS: The gene expression profile was determined by using cDNA expression arrays containing 588 cDNA. RESULTS: Aging and advanced glycation resulted in differential gene expression patterns of cerebrum and spleen compared with young mice. Among the 80 genes detected in cerebrum, 43 exhibited a change in mRNA ratios with aging or treatment. Thirty-four changes (79%) were common in aged and D-galactose treated mice, whereas the cerebrum from aged and AGE-lysine treated mice showed common changes in expression of 38 genes (88%). Of the 86 genes detected in spleen, 29 (34%) displayed an age-related decrease in expression, whereas 3 (3%) displayed an increase in expression levels with aging. Eighteen genes from the detectable genes exhibited expression changes in both cerebrum and spleen of mice. CONCLUSIONS: The gene expression profiles of D-galactose and AGE-lysine treated mice resemble those of aged mice. Use of cDNA hybridization arrays may provide a promising tool to explore the mechanism of aging at a molecular level.
