The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:709-714 (2005)
© 2005 The Gerontological Society of America
Effects of Human Na+/Dicarboxylate Cotransporter 3 on the Replicative Senescence of Human Embryonic Lung Diploid Fibroblasts
Xiangmei Chen,
Dongwei Cao,
Jianzhong Wang,
Li Yuan,
Zhe Feng,
Bo Fu,
Quan Hong,
Xiaojie Zhang,
Xueyuan Bai,
Yang Lu and
Rui Ding
Department of Nephrology, Kidney Center & Key Lab of PLA, General Hospital of PLA, Beijing, People's Republic of China.
Address correspondence to Xiangmei Chen, MD, PhD, Department of Nephrology, Kidney Center and Key Lab of PLA, General Hospital of PLA, Fuxing Road 28, Beijing 100853, People's Republic of China. E-mail: xmchen{at}public.bta.net.cn
 |
Abstract
|
|---|
To investigate the role of human Na+/dicarboxylate cotransporter 3 (hNaDC3) in the replicative senescence of normal human embryonic lung diploid fibroblasts (WI-38), a retroviral vector containing hNaDC3 was constructed. hNaDC3 was introduced into normal WI-38 cells through infection with the retroviral virus. Monoclones were selected with G418. The integration and expression of exotic genes were confirmed by Northern blot and Western blot. When compared with the control cells, WI-38 cells transfected with hNaDC3 cDNA showed significant suppression of growth rate (by 40%), increase of positive rate of SA-ß-gal staining, decrease of mitochondrial membrane potential, shortening of telomere length, and increase of P16 and P21 expression. The morphology characteristics of senescent fibroblasts appeared earlier. Our results have, for the first time, demonstrated that high expression of hNaDC3 may be able to, at least partly, promote the cellular senescence of human diploid fibroblasts.
PRIMARY mammal cells exhibit finite proliferative potential in vitro, which undergo a limited number of population doublings (PDs) before entering senescence in which they remain metabolically active but completely refractory to mitogenic stimuli (1). Cell senescence is considered to be the result of multiple factors (2). Energy metabolism is related to cell senescence, as food restriction may lengthen life span, reducing diseases and pathological changes concerning aging (3,4). NaDC is responsible for transporting the mediate products of Krebs cycle and is related to energy metabolism. For example, it provides 10%–15% energy of oxidizing metabolism for tubular epithelial cells in the kidney (5). In 2000, Rogina et al (6) found that mutation of Indy gene in the fruit fly lengthened its mean life span. Thirty-four percent of the products of Indy gene were the same as those of NaDC and 50% products were similar. Moreover Indy protein played a vital role in the energy metabolism. Recent findings manifested that ceNaDC1 and ceNaDC2 from Caenorhabditis elegans (C. elegans) showed significant sequence homology with the Indy gene, and corresponded at the functional level to the mammalian NaDC1 and NaDC3, respectively (7). The ceNaDC1 and ceNaDC2 genes were not expressed at the embryonic stage, but the expression was detectable all through the early larva stage to the adult stage. Independent function knockdown of these two transporters using the strategy of RNA interference suggested that ceNaDC1 was not associated with the regulation of average life span, whereas the knockdown of ceNaDC2 function led to a significant increase in the average life span. Disruption of the function of ceNaDC2 in C. elegans led to decreased availability of dicarboxylate to produce metabolic energy, thus creating a biological state similar to that of caloric restriction, and consequently leading to life span extension.
At present the effects of NaDC2 on life span have not been published yet. Here we hypothesized that ceNaDC2 is perhaps involved in the aging process by energy metabolism. In our experiment, hNaDC3, the counterpart of ceNaDC2, was introduced into normal human diploid fibroblast WI-38 cells through infection with retroviral virus, and the effects of hNaDC3 on replicative cellular senescence of WI-38 cells were examined.
|
METHODS
|
|---|
Cell Culture and Medium
Human embryonic lung diploid fibroblast WI-38 cells (obtained from the American Type Culture Collection) were grown in Earle's Minimum Essential Medium (GIBCO) with 10% heat-inactivated fetal bovine serum. GP2-293 cells were grown in Dulbecco's modified Eagle's medium with 10% bovine serum. All cells were grown in medium containing 100 µg/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO2.
Construction of Retroviral Vectors and Transfection of Human Diploid Fibroblasts
pLNCX2 was obtained from Clontech Inc. (CA, USA). The full-length hNaDC3 (2000 bp) was inserted into the retroviral vector pLNCX2-neo in both orientations. These two constructs were termed pLNCX2-hNaDC3 and pLNCX2-AShNaDC3. They were transfected with lipofectamine reagent (GIBCO) and vesicular stomatitis virus G protein (VSVG) into 5 x 105 GP2-293 cells (50%–60% confluence). The transfected GP2-293 cells were grown at 32°C in 5% CO2, and the medium containing retroviral virus was collected after 48–72 hours. hNaDC3 was then introduced into young human diploid fibroblast WI-38 cells (PD 17) (50%–60% confluence) through infection with retroviral virus. The transformants, after sustained selection by G418, were termed as WI-38/hNaDC3, WI-38/AShNaDC3, and WI-38/neo (as a control), respectively.
Northern and Western Blot
Total RNA was isolated from cells with a Trizol reagent kit. Samples (30 µg) of total cellular RNA were subjected to electrophoresis on denatured agarose gel, blotted onto nylon membrane (Hybond-N; GE Healthcare, NJ), and hybridized for 16 hours with hNaDC3 probe labeled with 
-32P-dCTP. 28S was used as the control. For Western blotting, 40 µg of denatured cell proteins were run on 10% and 15% sodium dodecyl sulfate-polyacrylamide gel, transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA), and probed with anti-hNaDC3 antibody, anti-p16, and anti-p21 monoclonal antibodies. The signals were detected with an enhanced chemiluminescence (ECL) system.
Measurement of PD
Transfected cell clones were selected with G418. The PD number of a clone grown to about 5 x 105 cells is approximately 19 from which the PD number was started to be counted. Therefore, the actual PD number of the transfected cells should be increased by 19 PD when compared with the untransfected cells (8).
Cell Growth Velocity by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] Assay
The amount 2.5 x 103 WI-38 cells per well were plated into 96-well plates. Ten microliters of MTT [3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (5 g/L) were added per well 4 hours ahead of measurement at the indicated time point. After 200 µl of dimethyl sulfoxide were added per well in the dark for 20 minutes, optical density (OD) values were obtained from a Microplate Reader (Bio-Rad) at the 492-nm wave.
Cell Cycle Analysis
The cells at 70%–80% confluence were washed with phosphate-buffered saline (PBS), detached with 0.25% trypsin, and fixed with 75% ethanol overnight, then were resuspended in 1 ml of PBS and stained with propidium iodide in the dark for 30 minutes. The DNA contents were measured with a fluorescence-activated cell sorting flow cytometry system (FACScan; BD Biosciences, San Jose, CA).
SA-ß-Gal Staining
After being washed in PBS and fixed for 5 minutes at room temperature in 3% formaldehyde, the cells were incubated overnight at 37°C (without CO2) with freshly prepared SA-ß-gal stain solution [1 mg/ml X-gal, 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2). To obtain the positive rate, six fields were chosen for each sample (9).
Measurement of Mitochondrial Membrane Potential (
)
When the cells, which had been planted into a 35-mm culture plate (MatTek, Ashland, MA), reached 70%–80% confluence, they were cultured in medium containing JC-1 at 10 µg/ml in the dark for 10 minutes at 37°C. Then cells were washed with Hanks solution. Confocal fluorescence microscopy was used to detect the changes of in normal WI-38 cells, pLNCX2-transfected, pLNCX2-hNaDC3-transfected, and pLNCX2-AShNaDC3-transfected WI-38 cells. Six fields were chosen to count cells with different colors (10).
Telomere Length Assay
A total of 10 µg of genomic DNA per sample was digested with restriction enzymes (11) Hinf I and Rsa I, subjected to electrophoresis on a 0.7% agarose gel, and transferred onto nylon membranes. The membrane was exposed in the ultraviolet instrument for 6 minutes and hybridized with a 5'-end [
-32P] ATP-labeled telomeric (TTAGGG)5 repeat probe. After hybridization for 24 hours, washing was carried out with 0.2 x SSC (sodium chloride sodium citric acid buffer) and 0.1% sodium dodecyl sulfate. The membrane was autoradiographed on Kodak X-ray film for 48–72 hours at –70°C. The image was analyzed using VDS Image software (Beijing University, P.R. China). The mean telomere restriction fragment was defined as follows: 
(ODi)/(ODi/Li), in which ODi is the densitometer output and Li is the length of the DNA at position i (11).
|
RESULTS
|
|---|
Integration of pLNCX2-hNaDC3 and pLNCX2-AShNaDC3 in WI-38 Cells
In our study, the full length of hNaDC3 cDNA was inserted in both orientations into the retroviral vector. The constructs pLNCX2-hNaDC3, pLNCX2-AShNaDC3, and pLNCX2, were transfected into early passage WI-38 cells, respectively. And G418-resistant cell clones were obtained with sustained drug selection. Northern blot and western blot showed that there was little expression of hNaDC3 mRNA and hNaDC3 protein in normal untransfected cells and in WI-38/neo cells. Both hNaDC3 mRNA and hNaDC3 protein were shown in WI-38/hNaDC3. However, in WI-38/AShNaDC3 cells, hNaDC3 mRNA was detected, but hNaDC3 protein was not detected (Figures 1 and 2).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 1. Integration of human Na+/dicarboxylate cotransporter 3 (hNaDC3) RNA by Northern blot. Lane 1: WI-38 cells; lane 2: WI-38/neo; lane 3: WI-38/hNaDC3; lane 4: WI-38/AShNaDC3. Northern blot showed exogenous hNaDC3 mRNA in the WI-38/hNaDC3 and WI-38/AShNaDC3 cells, but not in the WI-38/neo or untransfected WI-38 cells
|
|
View larger version (26K):
[in this window]
[in a new window]
|
Figure 2. Expression of human Na+/dicarboxylate cotransporter 3 (hNaDC3) protein by Western blot. Lane 1: WI-38 cells; lane 2: WI-38/neo; lane 3: WI-38/hNaDC3; lane 4: WI-38/AShNaDC3. Western blot showed the expression of hNaDC3 protein only in the WI-38/hNaDC3 and not in the WI-38/neo, WI-38/AShNaDC3, or WI-38 cells
|
|
Finite Extension of Proliferative Life Span in WI-38/AShNaDC3 Cells
WI-38 cells enter permanent growth arrest after completing a finite number of divisions. We observed the proliferative life span of WI-38/hNaDC3, WI-38/AShNaDC3, WI-38/neo, and untransfected cells at the same time. The results showed that the life spans of WI-38/AShNaDC3 (PD 48–51), WI-38/neo, and the normal untransfected cells were similar. In contrast, WI-38/hNaDC3 cells ceased cell division (PD 37–40) 10–12 PD earlier than the control cells.
Delay of Growth Inhibition by AShNaDC3
The WI-38/hNaDC3 showed nearly complete growth inhibition like senescent cells (PD 48), as the growing rate of WI-38/hNaDC3 cells (PD 37) was slower than that of WI-38/neo (PD 37), and similar to that of senescent cells (PD 48). The growing rates of WI-38/neo and WI-38/AShNaDC3 were similar to that of middle-aged WI-38 cells (PD 37) (Figure 3).
hASNaDC3 Postpones G1 Cell Arrest
The cell cycle profile was analyzed with flow cytometry (Table 1). WI-38/hNaDC3 cells had a percentage (80.2%) of cells (37 PD) at G1 phase similar to that (78.8%) of normal senescent WI-38 cells (48 PD). The results indicated that the G1 arrest was caused by hNaDC3 accumulation.
Cell Morphologic Changes and the SA-ß-Gal Senescence-Associated Marker
We monitored morphological changes after transfection. The WI-38/hNaDC3 cells (PD 37) showed increasing enlargement, flattening, and accumulation of cytoplasmic granular inclusions similar to those of the senescent cells (PD 48). However, the morphology of WI-38/neo and WI-38/AShNaDC3 cells (PD 37) was similar to that of the middle-aged WI-38 cells (PD 37). The specific senescence-associated marker, SA-ß-gal was assayed with X-gal staining. Only sporadic SA-ß-gal-positive cells were seen in young WI-38 cells (PD 20), whereas almost all of the WI-38/hNaDC3 cells were positively-stained just like the senescent cells (PD 48). The positive rates of WI-38/neo cells (PD 37) (26% ± 4%) and WI-38/AShNaDC3 cells (PD 37) (27% ± 3%) were similar to that of the middle-aged WI-38 cells (PD 37) (28% ± 3%) (Figure 4).
View larger version (82K):
[in this window]
[in a new window]
|
Figure 4. Morphology and SA-ß-gal staining for young WI-38 (PD 20) (A), middle-aged WI-38 (PD 37) (B), senescent WI-38 (PD 48) (C), WI-38/neo (PD 37) (D), WI-38/AShNaDC3 (PD 37) (E), and WI-38/hNaDC3 (PD 37) (F). Cells were microphotographed at a magnification of 10 x 10
|
|
Changes of Mitochondrial Membrane Potential
Approximately 95% of young cells (PD 20) showed red and orange fluorescence, and only a few cells showed green fluorescence. The rate of orange fluorescence cells had no significant differences among in the WI-38/AShNaDC3 (75% ± 4%), WI-38/neo (78% ± 3%), and middle-aged cells (75% ± 8.9%). The rate of green fluorescence in WI-38/hNaDC3 cells (63% ± 7.9%) was similar to that of the senescent cells (60% ± 8.9%), with no red fluorescence in either group (Figure 5). The above results indicated that hNaDC3 was able to enhance senescence in decreasing the mitochondrial membrane potential.
View larger version (38K):
[in this window]
[in a new window]
|
Figure 5. Changes of mitochondrial membrane potential with confocal fluorescence microscopy for young WI-38 (PD 20) (A), middle-aged WI-38 (PD 37) (B), senescent WI-38(PD 48) (C), WI-38/neo (PD 37) (D), WI-38/AShNaDC3 (PD 37) (E), and WI-38/hNaDC3 (PD 37) (F). Mitochondrial function was monitored by a fluorescent dye JC-1 to measure mitochondrial membrane potential. When loaded in the cells, JC-1 produces a green florescence at low membrane potential and red at high membrane potential. Cells were microphotographed at a magnification of x600
|
|
Inhibition of p16 and p21 by AShNaDC3
The expressions of p16 and p21 in WI-38/hNaDC3 cells (PD 37) were similar to those of senescent cells (PD 48), but were more than that of middle-aged WI-38 cells (p <.05). However, the expressions of p16 and p21 of WI-38/AShNaDC3 cells (PD 37) were similar to those of WI-38/neo (PD 37) cells. The expressions of p16 and p21 were lowest in young cells (PD 20) among all groups (p <.05). The results suggested that the expressions of p16 and p21 in normal WI-38 cells increased with aging, and hNaDC3 overexpression in cells was also able to increase the expressions of p16 and p21 (Figure 6, A–C).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 6. A, Western blot showing the expression of p16 and p21 for WI-38/hNaDC3 (PD 37) (lane 1), senescent WI-38 cells [population doubling time (PD) 48] (lane 2), WI-38/neo (PD 37) (lane 3), middle-aged WI-38 (PD 37) (lane 4), WI-38/AShNaDC3 (PD 37) (lane 5), and young WI-38 cells (PD 20) (lane 6); B, the comparisons of p16/ACTIN in six groups, *p <.05 versus the middle-aged WI-38 cells (PD 37); C, the comparisons of p21/ACTIN in six groups, *p <.05 versus the middle-aged WI-38 cells (PD 37)
|
|
hNaDC3 Slows Down Shortening of Telomere Terminal Restriction Fragment
Our results showed that the terminal restriction fragment (TRF) length of young cells was the longest among all the groups (p <.05) and that there were no significant differences in TRF length among WI-38/neo and WI-38/AShNaDC3 cells. However, the TRF length of WI-38/hNaDC3 cells, just like that of senescent cells, was shorter than that of WI-38/neo cells (Figure 7, A and B).
View larger version (53K):
[in this window]
[in a new window]
|
Figure 7. The mean terminal restriction fragment (TRF) length of telomere for six groups of WI-38 cells. A, M: marker ( DNA/HindIII); lane 1: young WI-38 cells [population doubling time (PD) 20]; lane 2: middle-aged WI-38 cells (PD 37); lane 3: WI-38/neo (PD 37); lane 4: WI-38/AShNaDC3 cells (PD 37); lane 5: senescent WI-38 cells (PD 48); lane 6: WI-38/hNaDC3 cells (PD 37); B, *p <.05 versus the middle-aged WI-38 cells (PD 37)
|
|
|
DISCUSSION
|
|---|
NaDC3 is in a family of transmembrane proteins that are responsible for transportation of intermediates of Krebs cycle, such as succinate, citrate, and -ketoglutarate. NaDC3 is involved in energy metabolism through Krebs cycle, so the expression and function of NaDC3 directly affect cell energetic metabolism (12, 13), which is associated with aging. In the 1980s, Weindruch and colleagues (14) reported that caloric absorption might have a significant effect on maximum life span of animals and that life span would be shortened when food amounts consumed exceeded the proper levels. In our laboratory, two polyclonal antibodies against hNaDC1 and hNaDC3 proteins were established and, with the two antibodies, the relationship between NaDC3 and aging was investigated (15, 16). We found that the expression of NaDC3 increased with aging in the Wistar rats, so we hypothesize that NaDC3 may be involved in the process of aging through energy metabolism.
It is not clear yet whether hNaDC3 can cause cell senescence. To answer this question, retroviral vectors were constructed and hNaDC3 cDNA was introduced into the young WI-38 cells. Substantial evidence has demonstrated that overexpression of hNaDC3 accelerated the aging of WI-38 cells. Mitochondria are responsible for metabolismtime of oxygen stress and aging (17,18), which has now been confirmed in our experiments.
Telomere length is considered as the clock of cell division and reflects cell division ability. Harley and colleagues (19) measured telomere length with TRF in 1990. Their result suggested that the cell telomere length came to be shortened little by little with the increase of cell mitosis. A fragment of TTAGGG will be lost with human somatic cell division. Cells will cease mitosis and enter into senescence when the telomere length cannot sustain its function. The gradual loss of telomere is a mechanism for senescence of the somatic cells. Normal somatic cell division in vivo shorten TRF by 15–40 bp every year, and TRF length of cultured cells in vitro will be shortened by 50–200 bp per PD. Von Zglinicki and colleagues (20) recently reported that accumulation of single-strand breaks by oxidative stress was the major cause of telomere shortening. H2O2 might induce cells to manifest senescent morphology and accelerate telomere shortening. So telomere length is not only a count machine for cell divisions but also a biologic marker of cell senescence.
With cell aging, cell divisions cease with G1 arrest and the expression of cell cycle proteins increases. We observed that the telomere length of WI-38/hNaDC3 was similar to that of the senescent cells. The results suggested that the high expression of hNaDC3 in cells shortened the TRF length of telomere. The cell cycle assay with FACS showed that both WI-38/hNaDC3 cells and aging cells were at G1 arrest. Western blot showed that hNaDC3 introduced into the WI-38 cells led to the increased expression of p16 and p21. All the results indicated that overexpression of hNaDC3 may accelerate the process of cell aging.
Both replicative senescence and stress-induced premature senescence are dependent on two major pathways (21). One is triggered by DNA damage and telomere damage and/or shortening, and involves the activation of p21 (waf-1) proteins. The second pathway results in the accumulation of p16 (Ink-4a) with the mitogen-activated protein kinase signaling pathway as the possible intermediate. We observed the decrease of mitochondrial membrane potential, the shortening of telomere length, and the increased expression of p16 and p21 in WI-38 cells transfected with hNaDC3. Our results suggested that the two senescence pathways can be involved in cell senescence through hNaDC3. It can be inferred that the following factors are involved in the acceleration of cell senescence, including loaded energetic metabolism after overexpression of hNaDC3: (a) increase of oxidative stress damage, and (b) increased metabolic products to enhance aging, such as telomere shortening and the increased expression of p16 and p21.
|
Acknowledgments
|
|---|
This study was supported by the Main State Basic Research Development Program of the People's Republic of China (PRC) (G2000057000), the Creative Research Group Fund from the National Foundation Committee of Natural Science of the PRC (30121005), and grants from the National Foundation Committee of Natural Science of the PRC (30270505 and 30370559).
|
Footnotes
|
|---|
Decision Editor: James R. Smith, PhD
Received October 29, 2004
Accepted January 19, 2005
|
References
|
|---|
- Hayflick L. The limited in vitro life time of human diploid cell strains. Exp Cell Res. 1965;37:614-636.[Medline]
- Tanjun Tong, Zongyu Zhang. The opportunity and challenge faced by molecular biology of aging in the new century. Chin J Geriatrics. 2000;19:167-169.
- Weindruch R. The retardation of aging by caloric restriction: studies in rodents and primates. Toxicol Pathol. 1996;24:742-745.[Medline]
- Eiam-Ong S, Eiam-Ong S, Sabatini S. Life-long food restriction prevents renal membrane lipid deposition and lowers renal work in rats. J Med Assoc Thai. 2001;84:(Suppl 1): S295-S305.
- Hamm LL. Renal handling of citrate. Kidney Int. 1990;38:728-735.[Medline]
- Rogina B, Reenan RA, Nilsen SP, et al. Extended life-span conferred by cotransporter gene mutations in drosophila. Science. 2000;290:2137-2140.[Abstract/Free Full Text]
- Fei YJ, Inoue K, Ganapathy V. Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span. J Biol Chem. 2003;278:6136-6144.[Abstract/Free Full Text]
- Duan J, Zhang Z, Tong T. Senescence delay of human diploid fibroblast induced by anti-sense p16INK4a expression. J Biol Chem. 2001;276:48325-48331.[Abstract/Free Full Text]
- Dimri GP, Lee X, Basile G, Acosta M, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92:9363-9367.[Abstract/Free Full Text]
- Xu M, Wang Y, Ayub A, Ashraf M. Mitochondrial K(ATP) channel activation reduces anoxic injury by restoring mitochondrial membrane potential. Am J Physiol. 2001;281:H1295-H303.
- Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature. 1990;345:458-460.[Medline]
- Simpson DP. Citrate excretion: a window on renal metabolism. Am J Physiol. 1983;244:F223-F234.
- Chen XM, Tsukaguchi H, Chen XZ, et al. Molecular and functional analysis of SDCT2, a novel rat sodium-dependent dicarboxylate transporter. J Clin Invest. 1999;103:1159-1168.[Medline]
- Weindruch R, Walford RL. Dietary restriction in mice beginning at one year of age: effect on life-span and spontaneous cancer incidence. Science. 1982;215:1415-1418.[Abstract/Free Full Text]
- He Y, Chen X, Yu Z, et al. Fusional expression and antibody preparation of human sodium-dependent dicarboxylate cotransporter 1 gene. Chin J Biochem Mol Biol. 2002;18:356-360.
- Wang J, Chen X, Zhu H, Peng L, Hong Q. Relationship between aging and renal high-affinity sodium-dependent dicarboxylate cotransporter-3 expression characterized with antifusion protein antibody. J Gerontol Biol Sci. 2003;58A:879-888.
- Sugrue MM, Tatton WG. Mitochondrial membrane potential in aging cells. Biol Signals Recept. 2001;10:176-188.[Medline]
- Ehrenberg B, Montana V, Wei MD, Wuskell JP, Loew LM. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J. 1988;53:785.[Abstract/Free Full Text]
- Harley CB, Futcher AB, Greider CW. Telomeres shorten during aging of human fibroblast. Nature. 1990;345:458-460.
- von Zglinicki V, Pilger R. Sitte N. Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic Biol Med. 2000;28:24-27.
- Toussaint O, Medrano EE, von Zglinicki T. Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Exp Gerontol. 2000;35:927-945.[Medline]
This article has been cited by other articles:
|
|
|
|
 
X.-Y. Bai, X. Chen, A.-Q. Sun, Z. Feng, K. Hou, and B. Fu
Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter
FASEB J,
August 1, 2007;
21(10):
2409 - 2417.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Methods
: young WI-38 [population doubling time (PD) 20]; 
: WI-38/neo (PD 37); 
: middle-aged WI-38 (PD 37); x: senescent WI-38 (PD 48); 
: WI-38/hNaDC3 (PD 37); •: WI-38/AShNaDC3 (PD 37). The WI-38/hNaDC3 showed nearly complete growth inhibition like senescent cells (PD 48). The growth rate or the growth potential of WI-38/neo and WI-38/AShNaDC3 was similar to the untransfected middle-aged cells (PD 37)