Two isomers of HDTIC isolated from Astragali Radix decrease the expression of p16 in 2BS cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Astragali Radix, the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1, 3] dioxolan-2, 6'-spirane-5', 6', 7', 8'-tetrahydro-indolizine-3'-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2 strongly delay replicative senescence of human fetal lung diploid fibroblasts (2BS). In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence.</p><p><b>METHODS</b>The effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants.</p><p><b>RESULTS</b>There was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings (PDs) grown from PD28 in 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of p16 was observed. The expression level of p21 increased with cell ageing. Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 micromol/L H2O2 for 5 minutes return to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 (1.0 micromol/L ) or HDTIC-2 (10 micromol/L ) for 1 hour.</p><p><b>CONCLUSIONS</b>Expression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16(INK4a)/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.</p>

Posttranscriptional induction of p21Waf1 mediated by ectopic p16INK4 in human diploid fibroblast / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Both p16(INK4) and p21(Waf1) are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16(INK4) could be activated by p21(Waf1) through transcriptional factor Sp1 in HeLa cells. This study was undertaken to determine the effects of p16(INK4) on the expression and functions of p21(Waf1).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>p16(INK4) may play an important role in the regulation of cellular senescence by modulating the p21(Waf1) protein level via the posttranscriptional mechanism.</p>

Aminoguanidine delays the replicative senescence of human diploid fibroblasts / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro.</p><p><b>METHODS</b>The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS).</p><p><b>RESULTS</b>Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD) and increased cumulative population doublings by at least 17 - 21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 micromol/L H(2)O(2) for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 micromol/L H(2)O(2) had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells.</p><p><b>CONCLUSION</b>Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, improvement of DNA repair ability and the slowdown of telomere shortening.</p>

Optimization of reporter gene assay: several factors influencing detection of promoter activity / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>Human embryonic lung fibroblast cells (2BS), HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined various 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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>