Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age‐related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi‐mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro‐inflammatory cytokines and extracellular matrix–degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi‐related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non‐senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.     

软骨细胞老化特征及机制的研究进展

软骨细胞的老化是一个极其复杂的过程，其特征包括：细胞不可逆的生长停滞于G1期；老化相关β-半乳糖苷酶的表达；端粒长度缩短；软骨细胞分化特征的改变。目前认为其机制为基因表达的程序性或减进性改变，包括肿瘤抑制基因p53和pRb等在细胞生长停滞中的作用；端粒结合蛋白和端粒酶对端粒长度的调节；细胞骨架蛋白的重组使软骨细胞形态的变化；基质降解酶类表达增加以及各种细胞因子的变化对软骨细胞代谢的影响。     

hnRNPU过表达抑制PIM1诱导的细胞衰老的机制研究

目的:探讨人前病毒整合位点1（PIM1）诱导细胞衰老的分子机制。方法:构建过表达PIM1的2BS细胞系,通过Western印迹法和衰老相关β-半乳糖苷酶染色实验测定过表达PIM1是否诱导细胞衰老。免疫沉淀联合质谱分析测定PIM1是否能有效免疫沉淀核异质核糖核蛋白U（hnRNPU）蛋白。运用Real-timePCR和Western印迹法测定PIM1是否影响hnRNPUmRNA和蛋白表达水平。构建同时过表达PIM1和hnRNPU的2BS细胞系,运用Western印迹法和衰老相关β-半乳糖苷酶染色实验检测hnNRPU过表达对PIM1诱导的细胞衰老的影响。结果:与空载对照组相比,过表达PIM1组中衰老信号通路关键基因p53、p21和p16的表达显著增加（p53,t=4.36,P<0.05;p21,t=3.814,P<0.05;p16,t=4.72,P<0.01）,衰老细胞的比例增加（t=6.831,P<0.01）。免疫沉淀联合质谱分析结果显示PIM1能有效免疫沉淀hnRNPU蛋白。PIM1过表达对hnRNPU的mRNA表达水平没有影响（t=0.295,P=0.783）,但是能抑制hnRNPU蛋白的表达（t=33.85,P<0.001）。与只过表达PIM1细胞相比,同时过表达PIM1和hnRNPU细胞,衰老信号通路关键基因p53、p21和p16的表达下降（p53,t=15.317,P<0.001;p21,t=8.012,P<0.01;p16,t=14.08,P<0.001）,衰老细胞的比例降低（t=10.38,P<0.01）。结论:hnRNPU过表达抑制PIM1诱导的细胞衰老。     

细胞衰老与p16INK4a的转录调控

抑癌基因p16~(INK4a)可特异地抑制CDK4及CDK6,在抑制细胞生长、促进细胞衰老等方面发挥重要的生物学作用。由于p16~(INK4a)功能的重要性,近年来,针对p16~(INK4a)转录调控方面的研究取得了一系列进展,发现了一系列正性和负性调控元件和转录调控因子,如:E47、Id1、Jun B、Bmi-1、RREB等,为进一步认识细胞增殖规律以及衰老进程具有重要的理论意义。     

Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.     

Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges

This study focuses on recent improvement in epithelial monolayer cultures originating from whole extirpated Botryllus schlosseri (Urochordata) buds. Buds (n = 2,000) were taken at different (A to D) blastogenic stages. We tested the suitability of 35 combinations of various substrates and media on attachment, cell spread, epithelial growth frequencies and on monolayer lifespans. Under favorable conditions, cultured buds at blastogenic stages B to D (but not stage A) started to attach to the substrates following a 3-day transient period that leads to formation of spheres and attached monolayers. Substrate type is important for the attachment and the development of monolayers. Under various culture conditions, some of stages B and C buds develop (3–20 days) one or more large (1 mm diameter) spheres. Stage D buds develop monolayers (up to 20% of buds) without going through a sphere phase. Neither spheres nor attached monolayers of epithelium were observed in stage A bud cultures. Spheres grew at a rate of 60 m in diameter per day using specific medium types and did not attach unless the appropriate substrate was present. When attached, epithelial monolayers expanded at a rate of 200 m in diameter per day, for 3–15 days, and subsequently detached and died. Sixteen types of media were tested. Medium and substrate combinations were found to determine epithelial lifespan. These results revealed significant improvements in the culture of epithelial monolayers from Botryllus palleal buds. However, an early senescence of the developed epithelial sheets (up to two weeks from onset of appearance) may indicate an internal ageing clock that should be taken into consideration in future approaches.     

Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss

The pathogenesis of progressive bile duct loss in primary biliary cirrhosis remains unclear. In this study, the involvement of cellular senescence of biliary epithelial cells was examined in liver tissue samples from patients with primary biliary cirrhosis (n = 33), and compared with control diseased and normal livers (n = 83). In addition, cellular senescence was induced by oxidative stress in cultured mouse biliary epithelial cells. Biliary epithelial cells in small bile ducts in primary biliary cirrhosis, especially those in patients presenting with chronic non-suppurative cholangitis, frequently expressed senescence-associated beta-galactosidase, and senescence-associated p16(INK4) and p21(WAF1/CIP). In contrast, senescence-associated markers were rarely expressed in small bile ducts in control livers. The infiltration of myeloperoxidase-positive inflammatory cells into biliary epithelial cell layers was closely associated with the cellular senescence of biliary epithelial cells in early-stage PBC. Cellular senescence of cultured mouse biliary epithelial cells was induced by treatment with H2O2 via the p38MAPK-dependent pathway and nitric oxide-augmented H2O2-induced cellular senescence. Oxidative stress- and nitric oxide-mediated cellular senescence may be involved in bile duct lesions, which are followed by progressive bile duct loss in primary biliary cirrhosis.     

Age‐ and site‐specific decline in insulin‐like growth factor‐I receptor expression is correlated with differential growth plate activity in the mouse hindlimb

The proximal and distal growth plates of the principal long bones do not contribute equally to longitudinal growth. Most forelimb elongation occurs at the shoulder and wrist, while most hindlimb growth occurs at the knee. This study examined whether insulin‐like growth factor‐I (IGF‐I), a potent growth regulator, could underlie this variation via differential receptor expression. The spatiotemporal distribution of the IGF‐I receptor (IGF‐IR) was mapped in hindlimb growth plates (overall and within regional zones) from immature mice using immunohistochemistry. Growth activity was assessed by size/morphology of the growth plate and proliferating cell nuclear antigen (PCNA) expression. Both IGF‐IR and PCNA staining declined considerably with age in the proximal femur and distal tibia (hip and ankle), but expression remained high in the more active distal femur and proximal tibia (knee) throughout growth. Growth plate size decreased with age in all sites, but the absolute and relative decline in IGF‐IR in the hips and ankles of older mice indicated a site‐specific loss of IGF‐I sensitivity in these less active regions. These results suggest that regulation of the IGF‐IR may at least partially mediate differential long bone growth, thereby providing a local mechanism for altering skeletal proportions absent modification of systemic hormone levels. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Oral senile amyloidosis in senescence accelerated mouse (SAM)

Oral senile amyloidosis in senescence accelerated mouse (SAM) was examined for two SAM sublines (P/2/Iw and R/1/Iw) and for various ages by light microscopy, immunohistochemistry, and electron microscopy. The amyloid deposition, identified by green birefrigence following Congo red stain, was observed only in P/2/Iw. In P/2/Iw, no amyloid deposition was found at age 6 months; however, frequency and extent of such deposits increased with advancing age. Distribution of amyloid deposition was as follows: along papillary layers of mucous epithelium in the tongue, the gingiva, the palate, and the buccal mucosa; foci in connective tissues; along vessels, muscles, and minor salivary glands. Immunohistochemically, the amyloid deposition was positive with anti-ASSAM serum being raised against a unique amyloid protein ASSAM which probably induced "senile systemic amyloidosis". P/2/Iw is useful as an animal model of oral senile amyloidosis.