染色质重塑是调控基因时序性表达的重要环节.衰老的人二倍体成纤维细胞核中有呈点状聚集的异染色质结构,这种特征性现象被称为衰老相关异染色质聚集(SAHF).K9M-H3和HP1是SAHF的标志性蛋白.在SAHF的形成过程中,p16INK4a/Rb途径和高迁移率蛋白A(high-mobility group A protein,HMGA protein)等许多因素起着非常重要的作用.最近研究表明,SAHF能够抑制E2F靶基因的表达,从而使细胞维持于稳定的衰老状态.SAHF的发现为细胞衰老的研究提供了一个新的生物学标志,并为细胞衰老状态的稳定维持提出了一种分子机制.
Background Both p16^INK4 and p21waf1 are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16^INK4 could be activated by p21waf1 through transcriptional factor Spl in HeLa cells. This study was undertaken to determine the effects of p16^INK4 on the expression and functions of p21^waf1. Methods Human diploid fibroblast 2BS cells were stably transfected with sense （2BS/p16^INK4）, antisense p16^INK4 （2BS/asp16^INK4） or empty vector （2BS/neo） .Then they were assayed by reverse-transcription polymerase chain reaction （RT-PCR）, fluorescence activated cell sorting （FACS） and Western blot. Results 2BS/p16^INK4 cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21^waf1 protein levels increased twofold in the 2BS/p16^INK4 cells, but not decreased in the 2BS/asp16^INK4 cells. P21^waf1 mRNA levels were not affected in neither 2BS/p16^INK4 nor 2BS/asp16^INK4 cells. Conclusion p16^INK4 may play an important role in the regulation of cellular senescence by modulating the p21^waf1 protein level via the posttranscriptional mechanism.
Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay. Methods Human embryonic lung fibroblast cells （2BS）, HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined vadous factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments. Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein （EGFP）. Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity. Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.
