Cloning and characterization of cellular senescence-associated genes in human fibroblasts by suppression subtractive hybridization

Cellular senescence marks the end of the proliferative life span of normal cells in tissue culture and occurs after cells have undergone a certain number of population doublings (PDLs). It is accompanied by alterations in the pattern of gene expression. A specific human embryonic lung diploid fibroblast cell line, 2BS, has been studied as a model of senescence in our laboratory. Here, we report a set of cellular senescence-associated genes identified from suppression subtractive cDNA libraries from senescent and young 2BS cells. They include three novel genes and six previously identified genes of unknown function. The genes whose functions are known belong to various functional pathways that have been reported to change with the onset of senescence. These include three pre-mRNA splicing factors with reduced expression in senescent cells, indicating that the regulation of mRNA splicing is altered during cell senescence. In addition, the expression of the gene TOM1 (target of Myb 1), which has not previously been associated with cellular senescence, is shown to increase in senescent cells, and we demonstrate that the expression of antisense TOM1 gene in 2BS cells can delay the progress of senescence.     

Telomerase modulates expression of growth-controlling genes and enhances cell proliferation

Most somatic cells do not express sufficient amounts of telomerase to maintain a constant telomere length during cycles of chromosome replication. Consequently, there is a limit to the number of doublings somatic cells can undergo before telomere shortening triggers an irreversible state of cellular senescence. Ectopic expression of telomerase overcomes this limitation, and in conjunction with specific oncogenes can transform cells to a tumorigenic phenotype. However, recent studies have questioned whether the stabilization of chromosome ends entirely explains the ability of telomerase to promote tumorigenesis and have resulted in the hypothesis that telomerase has a second function that also supports cell division. Here we show that ectopic expression of telomerase in human mammary epithelial cells (HMECs) results in a diminished requirement for exogenous mitogens and that this correlates with telomerase-dependent induction of genes that promote cell growth. Furthermore, we show that inhibiting expression of one of these genes, the epidermal growth factor receptor (EGFR), reverses the enhanced proliferation caused by telomerase. We conclude that telomerase may affect proliferation of epithelial cells not only by stabilizing telomeres, but also by affecting the expression of growth-promoting genes.     

Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro

Isolation of a true self-renewing stem cell from the human brain would be of great interest as a reliable source of neural tissue. Here, we report that human fetal cortical cells grown in epidermal growth factor expressed low levels of telomerase and telomeres in these cultures shortened over time leading to growth arrest after 30 weeks. Following leukemia inhibitory factor (LIF) supplementation, growth rates and telomerase expression increased. This was best demonstrated following cell cycle synchronization and staining for telomerase using immunocytochemistry. This increase in activity resulted in the maintenance of telomeres at approximately 7 kb for more than 60 weeks in vitro. However, all cultures displayed a lack of oligodendrotye production, decreases in neurogenesis over time and underwent replicative senescence associated with increased expression of p21 before 70 weeks in vitro. Thus, under our culture conditions, these cells are not stable, multipotent, telomerase expressing self-renewing stem cells. They may be more accurately described as human neural progenitor cells (hNPC) with limited lifespan and bi-potent potential (neurons/astrocytes). Interestingly, hNPC follow a course of proliferation, neuronal production and growth arrest similar to that seen during expansion and development of the human cortex, thus providing a possible model neural system. Furthermore, due to their high expansion potential and lack of tumorogenicity, these cells remain a unique and safe source of tissue for clinical transplantation.     

Cloning and expression of SAG: A novel marker of cellular senescence

Unlike immortalized cell lines, normal human fibroblasts in culture undergo replicative senescence in which the number of population doublings is limited. While fibroblasts display a variety of changes as they senesce in vitro, little is known about how gene expression varies as a function of population doubling level. We have used differential hybridization screening to identify human genes that are preferentially expressed in senescent cells. While we found several isolates that were up-regulated in late-passage cells, all appeared to be variants of the same cDNA, which we named senescence-associated gene (SAG). Our data show that SAG expression is threefold higher in senescent fibroblasts and closely parallels the progressive slowdown in growth potential, but is not cell-cycle regulated. Thus, SAG serves as an accurate marker for fibroblast growth potential during replicative senescence. Further studies demonstrated that SAG is a novel gene active in nearly all tissue types tested and that it is conserved through evolution. DNA sequencing data indicate that SAG contains a potential DNA-binding domain, suggesting that SAG may function as a regulatory protein.     

Human Umbilical Cord Matrix Stem Cells Maintain Multilineage Differentiation Abilities and Do Not Transform during Long-Term Culture

Umbilical cord matrix stem cells (UCMSC) have generated great interest in various therapeutic approaches, including liver regeneration. This article aims to analyze the specific characteristics and the potential occurrence of premalignant alterations of UCMSC during long-term expansion, which are important issues for clinical applications. UCMSC were isolated from the umbilical cord of 14 full-term newborns and expanded in vitro until senescence. We examined the long-term growth potential, senescence characteristics, immunophenotype and multilineage differentiation capacity of these cells. In addition, their genetic stability was assessed through karyotyping, telomerase maintenance mechanisms and analysis of expression and functionality of cell cycle regulation genes. The tumorigenic potential was also studied in immunocompromised mice. In vitro, UCMSC reached up to 33.7±2.1 cumulative population doublings before entering replicative senescence. Their immunophenotype and differentiation potential, notably into hepatocyte-like cells, remained stable over time. Cytogenetic analyses did not reveal any chromosomal abnormality and the expression of oncogenes was not induced. Telomere maintenance mechanisms were not activated. Just as UCMSC lacked transformed features in vitro, they could not give rise to tumors in vivo. UCMSC could be expanded in long-term cultures while maintaining stable genetic features and endodermal differentiation potential. UCMSC therefore represent safe candidates for liver regenerative medicine.     

Impact of IFN-β and LIF overexpression on human adipose-derived stem cells properties

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells that their therapeutic effects in various diseases make them an interesting tool in cell therapy. In the current study, we aimed to overexpress interferon-β (IFN-β) and leukemia inhibitory factor (LIF) cytokines in human ADSCs to evaluate the impact of this overexpression on human ADSCs properties. Here, we designed a construct containing IFN-β and LIF and then, transduced human adipose-derived stem cells (hADSCs) by this construct via a lentiviral vector (PCDH-513B). We assessed the ability of long-term expression of the transgene in transduced cells by western blot analysis and enzyme-linked immunosorbent assay techniques on Days 15, 45, and 75 after transduction. For the evaluation of stem cell properties, flow cytometry and differentiation assays were performed. Finally, the MTT assay was done to assess the proliferation of transduced cells compares to controls. Our results showed high-efficiency transduction with highest expression rates on Day 75 after transduction which were 70 pg/ml for IFN-β and 77.9 pg/ml for LIF in comparison with 25.60 pg/ml and 27.63 pg/ml, respectively, in untransduced cells (p = .0001). Also, transduced cells expressed a high level of ADSCs surface markers and successfully differentiated into adipocytes, chondrocytes, neural cells, and osteocytes besides the preservation rate of proliferation near untreated cells (p = .88). All in all, we successfully constructed an hADSC population stably overexpressed IFN-β and LIF cytokines. Considering the IFN-β and LIF anti-inflammatory and neuroprotective effects as well as immune-regulatory properties of hADSCs, the obtained cells of this study could be subjected for further evaluations in experimental autoimmune encephalomyelitis mice model.     

Retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells is potentiated by leukemia inhibitory factor

Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferation, survival and differentiation in a wide range of cell types. Here we show that LIF potentiates retinoic acid-mediated neural induction in pluripotent P19 embryonal carcinoma cells. This activity of LIF was demonstrated by a profounded neural morphology followed by increased expression of neural-specific proteins (N-CAM, III beta-tubulin, and GAP-43), up-regulation of early neural lineage-specific gene Mash-1, and down-regulation of early endoderm-specific genes -fetoprotein and GATA-4. Moreover, LIF also slows growth and increases the level of apoptosis in differentiating cells.     

Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan

It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins. These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential. We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth. Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced. At each passage cells were plated at densities sufficiently low that colony-forming efficiency could be calculated. We determined cumulative population doublings and also estimated the number of cell generations attained under each condition. FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan. The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used. The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical.     

The time-pattern of rises and falls in proliferation fades with senescence of mortal lines and is perpetuated in immortal rat hepatoma fao cell line

Summary Immortal cells perpetuate the rises and falls of proliferation that are progressively damped in mortal long-term cultured cells. For immortal rat hepatoma Fao cells, similar waves of proliferation occurred about every 3–4 wk. Under the same conditions, embryonic human fibroblasts and transformed but not immortalized embryonic fibroblasts display similarly recurring proliferation waves that progressively decrease in amplitude until senescence of the lines. In addition, strains of diploid normal human skin fibroblasts cultured under different culture conditions display a similar time-pattern of proliferation. Although the amplitude and baseline of these fluctuations are characteristic for each cell line, a common point was marked slow down in proliferation after every sequence of about 25 population doublings for all cells. Renewed proliferation waves of Fao cells allow about 22–23 additional population doublings each. Normal embryonic fibroblasts culture and its transformed counterpart accumulate about 30 and 60 population doublings, respectively, before senescence. Normal fibroblast strains accumulate about 25 population doublings over their entire life spans. This halt in proliferation after every stretch of about 25 population doublings may correspond to a structural or functional stop following attrition of telomeric DNA. This putative stop may be bypassed once in transformed embryonic cells and repetitively in immortal cells. In support of this hypothesis, we observed rapid telomere shortening, in two steps, during divisions of mortal embryonic cells, and maintenance of long telomeres in immortal Fao cells, which may indicate episodic repair of telomeres. Alternatively, such maintenance of long telomeres may reflect survival and successive clonal growth of rare cells with long telomeres. We suggest that the balance between telomere attribution and repair processes regulates the waves of proliferation. Equal contributors to these studies.     

Monitoring of cellular senescence by DNA-methylation at specific CpG sites

Replicative senescence has fundamental implications on cell morphology, proliferation, and differentiation potential. Here, we describe a simple method to track long-term culture based on continuous DNA-methylation changes at six specific CpG sites. This epigenetic senescence signature can be used as biomarker for various cell types to predict the state of cellular senescence with regard to the number of passages, population doublings, or days of in vitro culture.     

Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts.

The hic-5 gene encodes a novel protein with Zn finger-like (LIM) motifs, the expression of which increases during cellular senescence. The ectopic expression of hic-5 in nontumorigenic immortalized human fibroblasts, whose expression levels of hic-5 were significantly reduced in comparison with those of mortal cells, decreased colony-forming efficiency. Stable clones expressing high levels of hic-5 mRNA showed higher levels of mRNAs for several extracellular matrix-related proteins, along with the alteration of an alternative splicing as seen in senescent cells and decreased c-fos inducibility. Furthermore, these clones acquired a senescence-like phenotype, such as growth retardation; senescence-like morphology; and increased expression of Cip1/WAF1/sdi1 after 20 to 40 population doublings. On the other hand, antisense RNA expression of hic-5 in human normal diploid fibroblasts delayed the senescence process. HIC-5 was localized in nuclei and had affinity for DNA. Based on these observations, we speculated that HIC-5 affected the expression of senescence-related genes through interacting with DNA and thereby induced the senescence-like phenotypes. To our knowledge, hic-5 is the first single gene that could induce senescence-like phenotypes in a certain type of immortalized human cell and mediate the normal process of senescence.     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

Histone deacetylase inhibitors induce a senescence-like state in human cells by a p16-dependent mechanism that is independent of a mitotic clock

We show here that histone deacetylase inhibitors (HDACIs) sodium dibutyrate (SDB) and trichostatin A (TSA) induce a phenotype that has similarities to replicative senescence in human fibroblasts. There was no evidence that SDB accelerated a constitutive cell division counting mechanism as previously suggested because cells pretreated with SDB for three mean population doublings (MPDs) exhibited a similar overall proliferative life span to controls once SDB was withdrawn. SDB-treated cells upregulated the cell cycle inhibitors p21(WAF1) and p16(INK4A), but not p14(ARF), in the same sequential order as in senescence and the cells developed biochemical markers of senescence. However, the mechanism of senescence did not involve telomere dysfunction and there was no evidence for any posttranslational modification of p53. The expression of human papillomavirus (HPV) 16 E6 in human fibroblasts or targeted disruption of the p53 and p21(WAF) genes only weakly antagonized HDACI-induced senescence. However, expression of the E7 gene, which inhibits the function of pRb, cooperated with E6 to block SDB-induced senescence completely and human cells deficient in p16(INK4A) (but not p14(ARF)) were also resistant to SDB-induced senescence, suggesting that the p16(INK4A)/pRb pathway is the major mediator of HDACI-induced senescence in human cells. However, p53-/- mouse fibroblasts were resistant to HDACI-induced senescence, identifying p53 as the major pathway to senescence in this species.