Population Doubling Level Research Articles

Bisphenol A at environmentally relevant doses induces cyclooxygenase-2 expression and promotes invasion of human mesenchymal stem cells derived from uterine myoma tissue

ObjectiveUterine myoma is the most common benign reproductive tract tumor in women. Despite its high prevalence, the exact pathogenesis of these benign tumors remains unknown. Toward understanding the pathogenic mechanism of these tumors, we attempted to isolate human uterine myoma mesenchymal stem cells (hUM-MSCs), which may be the target cells for tumorigenesis. Furthermore, we tested the response of these hUM-MSCs to the environmental endocrine disruptor, bisphenol A (BPA), which may mimic the action of estrogen in hormone-sensitive organs such as the uterus. Materials and MethodsThe hUM-MSC lines were clonally derived from uterine myoma tissue using the MSU-1 medium supplemented with N-acetyl-l-cysteine and l-ascorbic acid-2-phosphate. These hUM-MSCs were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis for the expression of mesenchymal stem cell (MSC) surface markers (e.g., CD90 and CD105) and the transcription factor Oct-4. The proliferation potential was measured by the cumulative population doubling level and the colony-forming efficiency. ResultsPutative hUM-MSC lines expressed CD90, CD105, and the stem cell marker gene, Oct-4. The cells were capable of differentiating into adipocytes, osteoblasts, and chondrocytes. Bisphenol A treatment of these hUM-MSCs enhanced cell proliferation and colony-forming efficiency in a dose-responsive manner. At an environmentally relevant concentration (10−8 M), BPA moreover induced cyclooxygenase-2 (COX-2) gene expression and promoted cell migration and invasiveness. ConclusionThe hUM-MSC cell lines can be isolated from uterine myoma tissues. Bisphenol A could enhance cell proliferation and colony-forming efficiency, induce COX-2 gene expression, and promote migration and invasion of hUM-MSCs. The results imply that BPA has a detrimental effect on female health by promoting uterine tumorigenesis.

Exposure to High or Low Glucose Levels Accelerates the Appearance of Markers of Endothelial Cell Senescence and Induces Dysregulation of Nitric Oxide Synthase

To test the hypothesis that aging impairs endothelial cell response to glucose stress, we utilized a human umbilical vein endothelial cell in vitro model in which clinically relevant concentrations of normal (5.5 mM), high (25 mM), and low (1.5mM) glucose were tested. With advancing population doubling, exposure to normal glucose gradually decreased endothelial nitric oxide synthase expression and activity, resulting in slow, progressive development of markers of cell senescence (by population doubling level [PDL] 44). High or low glucose treatment accelerated the appearance of markers of senescence (by ~PDL 35) along with declines in endothelial nitric oxide synthase expression and activity. Human umbilical vein endothelial cells exposed to alternating low and high glucose gave even more rapid acceleration in the appearance of markers of senescence (by ~PDL 18) and reduction in endothelial nitric oxide synthase levels. Thus, exposure to low and high glucose induces earlier appearance of markers of endothelial cell senescence and dysregulation of the nitric oxide synthase gene and protein expression and function. These findings will help to elucidate endothelial dysfunction associated with glucose intolerance and improve future therapy for diabetic seniors.

Effects of serial passage on the characteristics and chondrogenic differentiation of canine umbilical cord matrix derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

A relationship exists between replicative senescence and cardiovascular health

A growing body of evidence demonstrates that the accumulation of senescent cells is a plausible ageing mechanism. It has been proposed that the senescence of vascular cells plays a causal role in the development of cardiovascular pathologies. A key prediction arising from this hypothesis is that cultures of cells derived from donors with cardiovascular disease will show reduced in vitro replicative capacities compared to those derived from disease-free controls. Accordingly, we carried out a formal review of the relationship among donor age, cardiovascular health status and maximum population doubling level attained in vitro by cultures of vascular smooth muscle and endothelial cells. Data were available to us on a total of 202 independent cell cultures. An inverse relationship was found to exist between replicative capacity and donor age in both endothelial and vascular smooth muscle cells. Cultures derived from donors with cardiovascular disease showed a lower overall replicative potential than age-matched healthy controls. In general the replicative potential at the start of the lifespan was found to be higher in those individuals without disease than those with disease and the difference in average cumulative population doublings (CPDs) in age-matched individuals in the two groups remained roughly constant throughout the lifetime. These results are consistent with the model in which the inherited replicative capacity of vascular cells is a stronger determinant of the onset of cardiovascular disease later in life, than wear-and-tear throughout the life course.

Isolation and characterization of equine amniotic membrane-derived mesenchymal stem cells

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco's modified Eagle's medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.

Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential.

Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15% FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11% than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11% is preferential for bMSC propagation.

Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine.

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAMα cells and induced to osteogenic status—their in vivo osteogenesis was subsequently investigated in rats. It was found that HAMα cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAMα cells. The expression of osteocalcin mRNA was increased in HAMα cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAMα cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy.

Isolation and Characterization of Canine Wharton's Jelly-Derived Mesenchymal Stem Cells

Wharton's jelly is a known stem cell source in humans. Because stem cells might provide a potential therapeutic role in canines, many stem cell sources are studied for isolation and characterization in the canine system. So far, there have been no reports identifying canine Wharton's jelly stem cells. In this study, we successfully isolated and characterized mesenchymal stem cells (MSCs) from canine Wharton's jelly. Canine Wharton's jelly-derived mesenchymal stem cells (cWJ-MSCs) that were grown in low-glucose DMEM medium have spindle-like shapes similar to human Wharton's jelly stem cells. We characterized the immunophenotypes of canine Wharton's jelly stem cells by FACS analysis and measured the cumulative population doubling level (CPDL). We investigated the differentiation of cWJ-MSCs with a trilineage differentiation assay to determine whether they were mesenchymal. Under various differentiation conditions, cWJ-MSCs presented chondrogenic, osteogenic, adipogenic, and neurogenic differentiation abilities in vitro. In conclusion, our results show that cWJ-MSCs might be a good source for stem cells. Furthermore, cWJ-MSCs might be useful as a cell therapy application for veterinary medicine.

Isolation and Characterization of Human Amniotic Mesenchymal Stem Cells and Their Chondrogenic Differentiation

Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stem cells (MSCs) because fHAM cells can barely survive to expand under culture conditions in vitro for a long time. In this study, we separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints. We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAMα cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen-DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor β3, transforming growth factor β3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAMα cells achieved 8-week survival in vivo. These results suggest that HAMα cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAMα cells could be an allotransplantable cell resource for cartilage repair.

Establishment of ultra long‐lived cell lines by transfection of TERT into normal human fibroblast TIG‐1 and their characterization

To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

Anti-aging effect of black rice against H2O2-induced premature senescence

This study was focused on the anti-aging effect of black rice under stress-induced premature senescence (SIPS) of WI-38 human diploid fibroblasts cells caused by hydrogen peroxide (H2O2), a well-established experimental model of cellular aging. The protective effect of the MeOH extract from black rice against H2O2-induced premature senescence was investigated using WI-38 cells by evaluating the cell viability, lipid peroxidation and cell life span. H2O2-treated WI-38 cells exhibited the phenomena of SIPS, the loss of cell viability, the increase of lipid peroxidation and shortening of the cell lifespan. However, the treatment with black rice attenuated cellular oxidative stress through increase in the cell viability and inhibition in lipid peroxidation. In addition, the life span of WI-38 cell showed extension of the population doubling level, suggesting that it would delay aging process. These results suggest that the MeOH extract from black rice may delay the aging process by attenuation of oxidative stress under SIPS cellular model. Key words: Black rice, ageing, WI-38 cell, premature senescence, oxidative stress.

Improvement of cell freezing technologies: towards a fully closed process

Cell culture from vials usually includes an open phase performed under laminar flow hoods. Even if disposable and small closed systems are available, at least the first cell culture step after vial thawing, which is a centrifugation, is performed in disposable but open containers. This step is critical as the risk of contamination is high and it also has a direct impact on process timelines. In order to reduce the risk of contamination during thawing, Merck Serono Biodevelopment studied the possibility of freezing cell banks in bags and to directly thawing them directly in disposable and closed containers. The first step of this study was to evaluate if the DMSO present in freezing media had to be removed just after vial thawing or not. In general, this cryoprotective organic solvent is removed by centrifugation to avoid any toxic effect on cells. So vials from two different mammalian cell lines were thawed, each with and without a centrifugation step to remove DMSO, or not. Afterwards the cell amplification was performed in the same way for the two different thawing procedures. The population doubling level and the viability were monitored for more than 15 days and there was no significant impact neither on cell growth nor on viability whether DMSO was removed or not just after thawing, or not. The important parameter to take into account was the percentage of DMSO after dilution which had to be less than 0,5%. In order to reduce the risk of preparing cell banks, Merck Serono Biodevelopment looked at different disposable closed systems which allow cell banks to be prepared without laminar flow. It is possible to expand cells in closed containers such as spinners, shake flasks or rollers with deep tubes in addition to bags or disposable bioreactors. In order to concentrate cells or to change the medium from cell amplification to a cell freezing medium a centrifugation step may be necessary. Single use 500mL centrifuge tubes with deep tube and air vent are also available. In order to freeze cells, Merck Serono Biodevelopment evaluated several bags from several suppliers and chose the NovaSeptum® 100mL bag and the NovaSeptum® Case. The combination of these 2 elements ensured the integrity of the bag during freezing at -80°C (Figure 1-2). Figure 1 NovaSeptum 100mL bag and NovaSeptum case configurations Figure 2 NovaSeptum 100mL bag and NovaSeptum case configurations Thus 50mL of two different concentrated mammalian cell suspensions were frozen at -80°C and thawed in disposable and closed containers. The cells were not centrifuged just after thawing but expanded in order to seed a production bioreactor. The cells were also maintained in order to see if there was a long term impact of non-DMSO removal on viability and cell growth. The population doubling level, the cell viability obtained in disposable closed cell culture containers in addition to viable cell density, metabolism, molecule quantity and quality obtained in bioreactors were monitored. These parameters were compared to the same process performed with cells coming from vials. There was no process performance difference obtained between cells frozen in bags and cells frozen in vials. Cell freezing in bags without DMSO removal allows a fully closed cell culture and production process to be performed. It also allows process timelines to be reduced by least 2 weeks. The study will continue with a focus on bags which are compatible with nitrogen containers. The quality aspect will also be studied in order to be able to prepare a full or part of manufacturing of cell banks in bags.

Systemic Delivery of Allogenic Muscle Stem Cells Induces Long-Term Muscle Repair and Clinical Efficacy in Duchenne Muscular Dystrophy Dogs

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

Cadaverine effect on microtubule polymerization

The effect of in vitro age and donor age on net release of adenosine and inosine was studied in cultures of normal human fibroblasts. Confluent cultures of low- (population doubling level [PDL] 23–25) and high- (PDL 43–45) passage human lung fibroblasts derived from a 16-week-old fetal donor (IMR-90) were incubated for 30 min in physiological saline and the release of adenosine and inosine into the saline was determined by HPLC. Release of adenosine and inosine into the saline bathing low-passage human skin fibroblasts derived from a 16-week-old fetal donor (GM6111) was also determined and compared with two strains of low-passage skin fibroblasts from aged (66–67 years) donors (GM3529 and GM3524). The release of adenosine and inosine by low-passage cultures of fetal lung fibroblasts was 911 and 225 pmol/30 min per mg protein, respectively. In high-passage cultures of lung fibroblasts, release of adenosine and inosine was significantly greater at 1403 and 351 pmol/30 min per mg protein, respectively. The release of adenosine and inosine by low-passage cultures of fetal skin fibroblasts was 250 and 179 pmol/30 min per mg protein, respectively. In low-passage skin fibroblasts from aged donors, release of adenosine and inosine was significantly greater at 583 and 652 pmol/30 min per mg protein, respectively. These results indicate that the net release of adenosine and inosine by cultured human fibroblasts into their extracellular environment is enhanced by in vitro aging of lung fibroblasts and is greater in skin fibroblasts from aged donors.

Paracrine Proliferative Signaling by Senescent Cells in World Health Organization Group 3 Pulmonary Hypertension

Paracrine Proliferative Signaling by Senescent Cells in World Health Organization Group 3 Pulmonary Hypertension

Increased Monocytic Adhesion by Senescence in Human Umbilical Vein Endothelial Cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.

Effects of 2-deoxyglucose and dehydroepiandrosterone on intracellular NAD+ level, SIRT1 activity and replicative lifespan of human Hs68 cells

2-Deoxy-D-glucose (2-DG) and dehydroepiandrosterone (DHEA) have been hypothesized to extend lifespan via mimicking calorie restriction (CR). Activation of sirtuins has been proposed to contribute to life extension of CR by increasing intercellular levels of NAD(+) in several organisms. However, it is unclear whether 2-DG and DHEA may affect intracellular NAD(+) levels and human sirtuin 1 (SIRT1) activities. Here, using human fibroblast Hs68 cells we showed that 2-DG increased intracellular NAD(+) levels in both time- and concentration-dependent manners. 2-DG also dose-dependently increased SIRT1 activities and the lifespan (measured as the cumulated growth curve of population doubling levels) of Hs68 cells. In contrast, DHEA at non-cytotoxic concentrations (≤50 μM) did not significantly affect NAD(+) levels, SIRT1 activities or the lifespan of Hs68 cells. These results suggest that 2-DG extends the lifespan of Hs68 cells by increased NAD(+) levels and SIRT1 activities, and that 2-DG has a potential as a CR mimetic.

The effect of aging on the DNA damage and repair capacity in 2BS cells undergoing oxidative stress.

Aging is associated with a reduction in the DNA repair capacity under oxidative stress. However, whether the DNA damage and repair capacity can be a biomarker of aging remains controversial. In this study, we demonstrated two cause-and-effect relationships, the one is between the DNA damage and repair capacity and the cellular age, another is between DNA damage and repair capacity and the level of oxidative stress in human embryonic lung fibroblasts (2BS) exposed to different doses of hydrogen peroxide (H2O2). To clarify the mechanisms of the age-related reduction in DNA damage and repair capacity, we preliminarily evaluated the expressions of six kinds of pivotal enzymes involved in the two classical DNA repair pathways. The DNA repair capacity was observed in human fibroblasts cells using the comet assay; the age-related DNA repair enzymes were selected by RT-PCR and then verified by Western blot in vitro. Results showed that the DNA repair capacity was negatively and linearly correlated with (i) cumulative population doubling (PD) levels only in the group of low concentration of hydrogen peroxide treatment, (ii) with the level of oxidative stress only in the group of young PD cells. The mRNA expression of DNA polymerase δ1 decreased substantially in senescent cells and showed negative linear-correlation with PD levels; the protein expression level was well consistent with the mRNA level. Taken together, DNA damage and repair capacity can be a biomarker of aging. Reduced expression of DNA polymerase δ1 may be responsible for the decrease of DNA repair capacity in senescent cells.