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Nicotinamide extends the replicative life span of primary human cells
Mechanisms of Ageing and Development 127 (2006) 511–514 www.elsevier.com/locate/mechagedev
Short communication
Nicotinamide extends the replicative life span of primary human cells Chang-Su Lim *, Malcolm Potts, Richard F. Helm Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA Accepted 7 February 2006 Available online 20 March 2006
Keywords: Nicotinamide; Fibroblasts; Replicative life span; Aging; Deacetylase
Aging and longevity in humans are regulated by genes as well as environmental factors (i.e., lifestyle). It is believed that antioxidants and vitamins, whether obtained naturally or through nutritional supplements, play a critical role in the aging process by prolonging normal cellular functions and inhibiting deleterious ones. While there is an apparent direct relationship between vitamin intake and human health, the relationship between vitamin intake and cellular aging remains unclear (Thomas, 2004). Here we show that nicotinamide (NAM) (a component of Vitamin B3) extends the replicative life span of primary human cells, in stark contrast to what has been reported for Saccharomyces cerevisiae. Nicotinamide and nicotinic acid in combination comprise Vitamin B3, otherwise known as niacin. The sirtuin (silent information regulator-2, Sir2) family of protein/histone deacetylases are nicotinamide adenine dinucleotide (NAD+)dependent enzymes of ancient origin. NAM is a product of sirtuin catalyzed deacetylation, and is also a natural noncompetitive inhibitor of Sir2p (yeast), Sir2alpha (mouse) and hSIRT1 (human) activity (Luo et al., 2001; Bitterman et al., 2002; Langley et al., 2002; Senawong et al., 2003; Blander and Guarente, 2004; Avalos et al., 2005). Sirtuin protein activity was associated directly with aging and longevity in a number of model organisms, and it was hypothesized that activation of hSIRT1 increases longevity in humans (Guarente and Kenyon, 2000; Imai et al., 2000; Tissenbaum and Guarente, 2001; Porcu and Chiarugi, 2005). NAM was shown to shorten the replicative life span of Saccharomyces cerevisiae by inhibiting Sir2p activity (Bitterman et al., 2002).
* Corresponding author. Tel.: +1 540 231 3799; fax: +1 540 231 7126. E-mail address: [email protected] (C.-S. Lim). 0047-6374/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2006.02.001
The NAM-mediated shortening of life span in yeast prompted us to test whether NAM also negatively regulated the replicative life span of human cells. Based on the known health benefits of vitamins in nutrition, we hypothesized that the role of NAM in life span regulation in humans and yeast may be distinctly different. Support for this hypothesis rests largely on the facts that the metabolic pathways involving the conversion of NAM to the redox cofactor NAD+ differ, and human cells do not accumulate lethal extrachromosomal rDNA circles. To test the hypothesis, we examined the replicative life span of primary human neonatal skin fibroblasts (82-6, a kind gift from Dr. Junko Oshima) in the presence and absence of NAM (0–10 mM; 1 M stock solution in distilled water and then filter-sterilized). It was shown that 82-6 cells senesce after approximately 40 population doublings (Krtolica et al., 2001). Cells (82-6) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; 4.5 g/l D-glucose) (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (v/v), 100 units/ml penicillin, and 100 mg/ml streptomycin in an incubator at 37 8C, 5% CO2 and 98% humidity. Approximately 125,000 cells were plated into a 75 mm2-flask and were trypsinized and counted when they reached 70–80% confluency. Population doublings versus time were plotted to determine the replicative life spans. Young to middle-aged 82-6 cells treated with 5– 10 mM of NAM had significantly extended replicative life span relative to the control (Fig. 1A). To examine whether NAM differentially affects cell proliferation of 82-6 cells with differing concentration of NAM (0–10 mM), cell proliferation assay was performed (Fig. 1B and C), revealing that 10 mM NAM is the most potent concentration leading to life span extension. To investigate whether NAM-mediated life span extension is common in other cell types, we used primary human embryonic lung fibroblasts (IMR-90), which were obtained from ATCC, Manassas, VA. To first determine the cytotoxicity of NAM in
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C.-S. Lim et al. / Mechanisms of Ageing and Development 127 (2006) 511–514
Fig. 1. NAM extends the replicative life span of primary human diploid somatic fibroblasts (82-6 and IMR-90). (A) Middle-aged primary human skin fibroblasts (826) were allowed to proliferate in the presence (0.5, 5, and 10 mM) or absence of NAM. Cell counts were performed at 70–80% confluency. Population doublings vs. time were plotted to determine the replicative life span. Samples were in duplicate and the average values of designated time points were plotted. (B) Cell proliferation assay of 82-6 cells. For cell proliferation measurements, cells were plated in duplicate at a density of 10,000 cells in 30 mm2 glass slide dishes and grown for 13 days and then fixed with 100% methanol for 5 min for imaging. Fixed 82-6 cells were then stained with 0.5% crystal violet dissolved in 50% methanol. Cells were extensively washed with ample distilled water three times and air-dried. Crystal violet was subsequently extracted with 10% acetic acid. Optical density (OD) measurements were performed at 562 nm to indicate the relative number of cells in each designated NAM treatment. Cell proliferation of 82-6 cells without NAM was defined as 1-fold. The relative cell proliferation in each NAM treatment was displayed in comparison with the control in fold, which correspond to each designated cellular images shown in figure. A representative of two independent experiments is shown. Average values between samples in duplicate are shown and the error bar shows a range of errors. (C) Transmitted light confocal images showing cell proliferation of age-matched 82-6 cells in the presence/absence of NAM [0 mM (PD42), 0.5 mM (PD42), 5 mM (PD43), and 10 mM (PD43)]. Scale bars, 100 mm. A Zeiss LSM 510 laser scanning microscope was used to capture cellular images (10). (D) Normal human embryonic fibroblasts (IMR-90) were allowed to proliferate in the presence (5 mM) or absence of NAM. Cells were counted at 70–80% confluency. Population doublings vs. time were plotted to determine the replicative life span. This is a representative of three independent life span assays. (E) Transcriptional activity of p53 is increased in the presence of hSIRT1-H363Y. Transient transfection assays were carried out, which revealed that hSIRT1-H363Y functions as a dominant negative for p53 transcriptional activity due to its failure to inactivate p53 by deacetylation. (1) Saos-2 cells were transfected with p53 reporter (b-galactosidase, p53RE) DNA (a generous gift of Dr. Koji Itahana) and internal transfection control (luciferase, CMV-Luc); (2) cells were transfected with p53RE, CMV-Luc and p53; (3) cells were transfected p53RE, CMV-luc, p53 and hSIRT1; (4) cells were transfected with p53RE, CMV-Luc, p53 and hSIRT1-H363Y. Transient transfections were performed by plating Saos-2 cells at a density of 200,000 cells per well in 6 well plates 24 h before transfection using FuGene 6 (Roche Diagnostics, Indianapolis, IN) according to manufacturer’s instructions. 72 h after transfection, luciferase assay was done using the luciferase assay kit (Promega, Madison, WI), and luciferase activity was used to normalize transfection efficiency. To measure p53 transcriptional activity, b-galactosidase activity was measured using the Galacto-StarTM b-galactosidase reporter gene assay system (Applied Biosystems, Foster City, CA). X-axis denotes designated transfections, and Y-axis shows p53 transcriptional activity as fold induction (b galactosidase activity) compared to control (1-fold). A representative of four independent transient transfections is shown. Each independent transfection was in duplicate. Average values of duplicate samples are shown and the error bar shows a range of errors. (F) A dominant negative form of hSIRT1 (H363Y) provided no change in the replicative life span of 82-6 cells. The full length hSIRT1 cDNA was obtained from Dr. Fuyuki Ishikawa (University of Kyoto, Japan). The EcoRI and BamHI fragment of hSIRT1 was inserted into the EcoRI and BamHI sites of the pLXSN vector (Clontech, Mountain View, CA). The QuikChange II-XL Site-Directed Mutagenesis Kit (Stratagene, Carlsbad, CA) was used to introduce a point mutation at amino acid 363 (histidine to tyrosine) according to the manufacturer’s instructions. The sequences of PCR primers used in this mutagenesis are available upon request. To generate a retroviral stock (pLXSN and pLXSN-hSIRT1-H363Y), RetroPackTM PT67 packaging cells (Clontech, Mountain View, CA) were cultured according to the manufacturer’s instructions. DNAs (pLXSN and pLXSN-hSIRT1-H363Y) were transfected transiently into this retrovirus packaging cell line following the manufacturer’s instructions. After selection for infected cells using the antibiotic G418, the virus infection efficiencies were determined by comparing the relative number of cells between mock-infected control cells grown with and without G418. Thereafter, 500,000 infected cells of each cell strain were plated into 150 mm2flasks. (G) Verification of hSIRT1-H363Y overexpression in 82-6 cells collected from the final cell count during the course of life span assays. 100 mg of whole cell extracts (WCE) were used followed by immunoblotting using an anti-hSIRT1 antibody (Santa Cruz Biotech, Santa Cruz, CA). Anti-tubulin was used as a protein loading control.
C.-S. Lim et al. / Mechanisms of Ageing and Development 127 (2006) 511–514
IMR-90 cells, we performed cell proliferation assay with 0– 10 mM NAM over 9 weeks. It was found that 5 mM NAM was the most effective concentration for cell proliferation of IMR90 cells while 10 mM NAM was rather toxic (not shown). Therefore we performed the life span assays with IMR-90 cells in the presence and absence of NAM (0 and 5 mM), supplemented fresh every five days. In this case 5 mM NAM also extends the replicative life span of IMR-90 cells (Fig. 1D). Taken together, our findings suggest that NAM-mediated life span extension may be a common phenomenon in some types of primary mammalian cells. Our data support the hypothesis that the role(s) of NAM in the replicative life span of yeast and human cells are fundamentally different. As cell culture concentrations of NAM on the order 5 mM were shown to abolish the endogenous hSIRT1 activity in mammalian cells (Bitterman et al., 2002; Langley et al., 2002), the NAM-mediated life span extension process observed appears to be independent of hSIRT1 activity. It was previously shown that the hSIRT1-H363Y, which lacks the deacetylase activity of hSIRT1, functions in a dominant negative fashion for p53 deacetylase activity, probably due to its competitive binding to p53 as efficiently as the endogenous wild type hSIRT1, resulting in increase in p53 acetylation when overexpressed (Vaziri et al., 2001; Langley et al., 2002). In order to explore further whether replicative life span is mediated in a hSIRT1-independent manner, we blocked endogenous hSIRT1 activity in 82-6 cells through introduction of a dominant negative form of hSIRT1 (H363Y). The hSIRT1-H363Y was constructed by substituting tyrosine for histidine at amino acid 363 located in the deacetylase catalytic domain of hSIRT1, and verified by transient transfection assays (Fig. 1E). Subsequent overexpression provided no change in the replicative life span of 82-6 cells (Fig. 1F and G). As it has previously been shown that hSIRT1-mediated acetylation status of p53 does not contribute to replicative life span regulation (Michishita et al., 2005), we hypothesize that the action of NAM is independent of the deacetylase activity of hSIRT1. This observation is consistent with previous findings that the life span of a C. elegans sirtuin knock out mutant (Sir2.1, R11A8.4) was similar to that of control animals (C.-S. L., unpublished results). Our findings are further supported by two recent reports that replicative life span of mammalian cells (human and mouse) is likely to be independent of SIRT1 activity (Chua et al., 2005; Michishita et al., 2005). We propose that the NAM-mediated life span extension in human cells is largely an hSIRT1-independent process (Kaeberlein et al., 2004). However, we cannot exclude the possibility that there are other hSIRT1-dependent processes that do not rely upon its deacetylase activity (e.g., protein binding partners). While NAM was shown previously to provide a rapid and temporary reversal of the aging phenotype of human cells (Matuoka et al., 2001), life span extension was not observed. This may be due to the use of older cells for the NAM-mediated life span extension assays. At least for the cell types (82-6 and IMR-90) investigated in the present report, NAM is clearly beneficial to replicative life span of different
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types of primary human cells in monolayer culture leading to life span extension. The further development, advancement and modification of robust cellular aging hypotheses will require information on the metabolic fate of NAM, and an understanding of the quantitative contribution of hSIRT1 to life span extension in human cells. As the NAM-mediated life span extension described here is proposed to be largely independent of hSIRT1 deacetylase activity, and possibly age-dependent, efforts should concentrate on dissecting the mechanisms of age-dependent NAM-mediated processes at the cellular and molecular levels. Such investigations will lead to a more holistic picture of aging and life span extension, as well as a better understanding of the pleiotropic effects of NAM in human health. Acknowledgments This was in part supported by the Multidisciplinary University Research Initiative (MURI) program of the Department of Defense (Biomimetic Cell and Tissue Stasis; N00014-01-1-0852), the Long Term Storage of Blood Products Program of DARPA (N00173-98-1-G005-P00004 and N0017302-1-G0 16), and a seed grant from the VT/UVA/Carilion Biomedical Institute Research Initiative (C.-S. L.). References Avalos, J.L., Bever, K.M., Wolberger, C., 2005. Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol. Cell 17, 855–868. Bitterman, K.J., Anderson, R.M., Cohen, H.Y., Latorre-Esteves, M., Sinclair, D.A., 2002. Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J. Biol. Chem. 277, 45099–45107. Blander, G., Guarente, L., 2004. The Sir2 family of protein deacetylases. Annu. Rev. Biochem. 73, 417–435. Chua, K.F., Mostoslavsky, R., Lombard, D.B., Pang, W.W., Saito, S., Franco, S., Kaushal, D., Cheng, H.L., Fischer, M.R., Stokes, N., Murphy, M.M., Appella, E., Alt, F.W., 2005. Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress. Cell Metab. 2, 67–76. Guarente, L., Kenyon, C., 2000. Genetic pathways that regulate ageing in model organisms. Nature 408, 255–262. Imai, S., Armstrong, C.M., Kaeberlein, M., Guarente, L., 2000. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800. Kaeberlein, M., Kirkland, K.T., Fields, S., Kennedy, B.K., 2004. Sir2-independent life span extension by calorie restriction in yeast. PLoS Biol. 2, e296. Krtolica, A., Parrinello, S., Lockett, S., Desprez, P., Campisi, J., 2001. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging. Proc. Natl. Acad. Sci. U.S.A. 98, 12072–12077. Langley, E., Pearson, M., Faretta, M., Bauer, U.M., Frye, R.A., Minucci, S., Pelicci, P.G., Kouzarides, T., 2002. Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. Embo J. 21, 2383– 2396. Luo, J., Nikolaev, A.Y., Imai, S., Chen, D., Su, F., Shiloh, A., Guarente, L., Gu, W., 2001. Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 107, 137–148. Matuoka, K., Chen, K.Y., Takenawa, T., 2001. Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation. Cell Mol. Life Sci. 58, 2108–2116.
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Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C., Horikawa, I., 2005. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol. Biol. Cell 16, 4623–4635. Porcu, M., Chiarugi, A., 2005. The emerging therapeutic potential of sirtuininteracting drugs: from cell death to lifespan extension. Trends Pharmacol. Sci. 26, 94–103. Senawong, T., Peterson, V.J., Avram, D., Shepherd, D.M., Frye, R.A., Minucci, S., Leid, M., 2003. Involvement of the histone deacetylase SIRT1 in chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting
protein 2-mediated transcriptional repression. J. Biol. Chem. 278, 43041– 43050. Thomas, D.R., 2004. Vitamins in health and aging. Clin. Geriatr. Med. 20, 259– 274. Tissenbaum, H.A., Guarente, L., 2001. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410, 227–230. Vaziri, H., Dessain, S.K., Ng Eaton, E., Imai, S.I., Frye, R.A., Pandita, T.K., Guarente, L., Weinberg, R.A., 2001. hSIR2 (SIRT1) functions as an NADdependent p53 deacetylase. Cell 107, 149–159.
Short communication
Nicotinamide extends the replicative life span of primary human cells Chang-Su Lim *, Malcolm Potts, Richard F. Helm Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA Accepted 7 February 2006 Available online 20 March 2006
Keywords: Nicotinamide; Fibroblasts; Replicative life span; Aging; Deacetylase
Aging and longevity in humans are regulated by genes as well as environmental factors (i.e., lifestyle). It is believed that antioxidants and vitamins, whether obtained naturally or through nutritional supplements, play a critical role in the aging process by prolonging normal cellular functions and inhibiting deleterious ones. While there is an apparent direct relationship between vitamin intake and human health, the relationship between vitamin intake and cellular aging remains unclear (Thomas, 2004). Here we show that nicotinamide (NAM) (a component of Vitamin B3) extends the replicative life span of primary human cells, in stark contrast to what has been reported for Saccharomyces cerevisiae. Nicotinamide and nicotinic acid in combination comprise Vitamin B3, otherwise known as niacin. The sirtuin (silent information regulator-2, Sir2) family of protein/histone deacetylases are nicotinamide adenine dinucleotide (NAD+)dependent enzymes of ancient origin. NAM is a product of sirtuin catalyzed deacetylation, and is also a natural noncompetitive inhibitor of Sir2p (yeast), Sir2alpha (mouse) and hSIRT1 (human) activity (Luo et al., 2001; Bitterman et al., 2002; Langley et al., 2002; Senawong et al., 2003; Blander and Guarente, 2004; Avalos et al., 2005). Sirtuin protein activity was associated directly with aging and longevity in a number of model organisms, and it was hypothesized that activation of hSIRT1 increases longevity in humans (Guarente and Kenyon, 2000; Imai et al., 2000; Tissenbaum and Guarente, 2001; Porcu and Chiarugi, 2005). NAM was shown to shorten the replicative life span of Saccharomyces cerevisiae by inhibiting Sir2p activity (Bitterman et al., 2002).
* Corresponding author. Tel.: +1 540 231 3799; fax: +1 540 231 7126. E-mail address: [email protected] (C.-S. Lim). 0047-6374/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2006.02.001
The NAM-mediated shortening of life span in yeast prompted us to test whether NAM also negatively regulated the replicative life span of human cells. Based on the known health benefits of vitamins in nutrition, we hypothesized that the role of NAM in life span regulation in humans and yeast may be distinctly different. Support for this hypothesis rests largely on the facts that the metabolic pathways involving the conversion of NAM to the redox cofactor NAD+ differ, and human cells do not accumulate lethal extrachromosomal rDNA circles. To test the hypothesis, we examined the replicative life span of primary human neonatal skin fibroblasts (82-6, a kind gift from Dr. Junko Oshima) in the presence and absence of NAM (0–10 mM; 1 M stock solution in distilled water and then filter-sterilized). It was shown that 82-6 cells senesce after approximately 40 population doublings (Krtolica et al., 2001). Cells (82-6) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; 4.5 g/l D-glucose) (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (v/v), 100 units/ml penicillin, and 100 mg/ml streptomycin in an incubator at 37 8C, 5% CO2 and 98% humidity. Approximately 125,000 cells were plated into a 75 mm2-flask and were trypsinized and counted when they reached 70–80% confluency. Population doublings versus time were plotted to determine the replicative life spans. Young to middle-aged 82-6 cells treated with 5– 10 mM of NAM had significantly extended replicative life span relative to the control (Fig. 1A). To examine whether NAM differentially affects cell proliferation of 82-6 cells with differing concentration of NAM (0–10 mM), cell proliferation assay was performed (Fig. 1B and C), revealing that 10 mM NAM is the most potent concentration leading to life span extension. To investigate whether NAM-mediated life span extension is common in other cell types, we used primary human embryonic lung fibroblasts (IMR-90), which were obtained from ATCC, Manassas, VA. To first determine the cytotoxicity of NAM in
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C.-S. Lim et al. / Mechanisms of Ageing and Development 127 (2006) 511–514
Fig. 1. NAM extends the replicative life span of primary human diploid somatic fibroblasts (82-6 and IMR-90). (A) Middle-aged primary human skin fibroblasts (826) were allowed to proliferate in the presence (0.5, 5, and 10 mM) or absence of NAM. Cell counts were performed at 70–80% confluency. Population doublings vs. time were plotted to determine the replicative life span. Samples were in duplicate and the average values of designated time points were plotted. (B) Cell proliferation assay of 82-6 cells. For cell proliferation measurements, cells were plated in duplicate at a density of 10,000 cells in 30 mm2 glass slide dishes and grown for 13 days and then fixed with 100% methanol for 5 min for imaging. Fixed 82-6 cells were then stained with 0.5% crystal violet dissolved in 50% methanol. Cells were extensively washed with ample distilled water three times and air-dried. Crystal violet was subsequently extracted with 10% acetic acid. Optical density (OD) measurements were performed at 562 nm to indicate the relative number of cells in each designated NAM treatment. Cell proliferation of 82-6 cells without NAM was defined as 1-fold. The relative cell proliferation in each NAM treatment was displayed in comparison with the control in fold, which correspond to each designated cellular images shown in figure. A representative of two independent experiments is shown. Average values between samples in duplicate are shown and the error bar shows a range of errors. (C) Transmitted light confocal images showing cell proliferation of age-matched 82-6 cells in the presence/absence of NAM [0 mM (PD42), 0.5 mM (PD42), 5 mM (PD43), and 10 mM (PD43)]. Scale bars, 100 mm. A Zeiss LSM 510 laser scanning microscope was used to capture cellular images (10). (D) Normal human embryonic fibroblasts (IMR-90) were allowed to proliferate in the presence (5 mM) or absence of NAM. Cells were counted at 70–80% confluency. Population doublings vs. time were plotted to determine the replicative life span. This is a representative of three independent life span assays. (E) Transcriptional activity of p53 is increased in the presence of hSIRT1-H363Y. Transient transfection assays were carried out, which revealed that hSIRT1-H363Y functions as a dominant negative for p53 transcriptional activity due to its failure to inactivate p53 by deacetylation. (1) Saos-2 cells were transfected with p53 reporter (b-galactosidase, p53RE) DNA (a generous gift of Dr. Koji Itahana) and internal transfection control (luciferase, CMV-Luc); (2) cells were transfected with p53RE, CMV-Luc and p53; (3) cells were transfected p53RE, CMV-luc, p53 and hSIRT1; (4) cells were transfected with p53RE, CMV-Luc, p53 and hSIRT1-H363Y. Transient transfections were performed by plating Saos-2 cells at a density of 200,000 cells per well in 6 well plates 24 h before transfection using FuGene 6 (Roche Diagnostics, Indianapolis, IN) according to manufacturer’s instructions. 72 h after transfection, luciferase assay was done using the luciferase assay kit (Promega, Madison, WI), and luciferase activity was used to normalize transfection efficiency. To measure p53 transcriptional activity, b-galactosidase activity was measured using the Galacto-StarTM b-galactosidase reporter gene assay system (Applied Biosystems, Foster City, CA). X-axis denotes designated transfections, and Y-axis shows p53 transcriptional activity as fold induction (b galactosidase activity) compared to control (1-fold). A representative of four independent transient transfections is shown. Each independent transfection was in duplicate. Average values of duplicate samples are shown and the error bar shows a range of errors. (F) A dominant negative form of hSIRT1 (H363Y) provided no change in the replicative life span of 82-6 cells. The full length hSIRT1 cDNA was obtained from Dr. Fuyuki Ishikawa (University of Kyoto, Japan). The EcoRI and BamHI fragment of hSIRT1 was inserted into the EcoRI and BamHI sites of the pLXSN vector (Clontech, Mountain View, CA). The QuikChange II-XL Site-Directed Mutagenesis Kit (Stratagene, Carlsbad, CA) was used to introduce a point mutation at amino acid 363 (histidine to tyrosine) according to the manufacturer’s instructions. The sequences of PCR primers used in this mutagenesis are available upon request. To generate a retroviral stock (pLXSN and pLXSN-hSIRT1-H363Y), RetroPackTM PT67 packaging cells (Clontech, Mountain View, CA) were cultured according to the manufacturer’s instructions. DNAs (pLXSN and pLXSN-hSIRT1-H363Y) were transfected transiently into this retrovirus packaging cell line following the manufacturer’s instructions. After selection for infected cells using the antibiotic G418, the virus infection efficiencies were determined by comparing the relative number of cells between mock-infected control cells grown with and without G418. Thereafter, 500,000 infected cells of each cell strain were plated into 150 mm2flasks. (G) Verification of hSIRT1-H363Y overexpression in 82-6 cells collected from the final cell count during the course of life span assays. 100 mg of whole cell extracts (WCE) were used followed by immunoblotting using an anti-hSIRT1 antibody (Santa Cruz Biotech, Santa Cruz, CA). Anti-tubulin was used as a protein loading control.
C.-S. Lim et al. / Mechanisms of Ageing and Development 127 (2006) 511–514
IMR-90 cells, we performed cell proliferation assay with 0– 10 mM NAM over 9 weeks. It was found that 5 mM NAM was the most effective concentration for cell proliferation of IMR90 cells while 10 mM NAM was rather toxic (not shown). Therefore we performed the life span assays with IMR-90 cells in the presence and absence of NAM (0 and 5 mM), supplemented fresh every five days. In this case 5 mM NAM also extends the replicative life span of IMR-90 cells (Fig. 1D). Taken together, our findings suggest that NAM-mediated life span extension may be a common phenomenon in some types of primary mammalian cells. Our data support the hypothesis that the role(s) of NAM in the replicative life span of yeast and human cells are fundamentally different. As cell culture concentrations of NAM on the order 5 mM were shown to abolish the endogenous hSIRT1 activity in mammalian cells (Bitterman et al., 2002; Langley et al., 2002), the NAM-mediated life span extension process observed appears to be independent of hSIRT1 activity. It was previously shown that the hSIRT1-H363Y, which lacks the deacetylase activity of hSIRT1, functions in a dominant negative fashion for p53 deacetylase activity, probably due to its competitive binding to p53 as efficiently as the endogenous wild type hSIRT1, resulting in increase in p53 acetylation when overexpressed (Vaziri et al., 2001; Langley et al., 2002). In order to explore further whether replicative life span is mediated in a hSIRT1-independent manner, we blocked endogenous hSIRT1 activity in 82-6 cells through introduction of a dominant negative form of hSIRT1 (H363Y). The hSIRT1-H363Y was constructed by substituting tyrosine for histidine at amino acid 363 located in the deacetylase catalytic domain of hSIRT1, and verified by transient transfection assays (Fig. 1E). Subsequent overexpression provided no change in the replicative life span of 82-6 cells (Fig. 1F and G). As it has previously been shown that hSIRT1-mediated acetylation status of p53 does not contribute to replicative life span regulation (Michishita et al., 2005), we hypothesize that the action of NAM is independent of the deacetylase activity of hSIRT1. This observation is consistent with previous findings that the life span of a C. elegans sirtuin knock out mutant (Sir2.1, R11A8.4) was similar to that of control animals (C.-S. L., unpublished results). Our findings are further supported by two recent reports that replicative life span of mammalian cells (human and mouse) is likely to be independent of SIRT1 activity (Chua et al., 2005; Michishita et al., 2005). We propose that the NAM-mediated life span extension in human cells is largely an hSIRT1-independent process (Kaeberlein et al., 2004). However, we cannot exclude the possibility that there are other hSIRT1-dependent processes that do not rely upon its deacetylase activity (e.g., protein binding partners). While NAM was shown previously to provide a rapid and temporary reversal of the aging phenotype of human cells (Matuoka et al., 2001), life span extension was not observed. This may be due to the use of older cells for the NAM-mediated life span extension assays. At least for the cell types (82-6 and IMR-90) investigated in the present report, NAM is clearly beneficial to replicative life span of different
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types of primary human cells in monolayer culture leading to life span extension. The further development, advancement and modification of robust cellular aging hypotheses will require information on the metabolic fate of NAM, and an understanding of the quantitative contribution of hSIRT1 to life span extension in human cells. As the NAM-mediated life span extension described here is proposed to be largely independent of hSIRT1 deacetylase activity, and possibly age-dependent, efforts should concentrate on dissecting the mechanisms of age-dependent NAM-mediated processes at the cellular and molecular levels. Such investigations will lead to a more holistic picture of aging and life span extension, as well as a better understanding of the pleiotropic effects of NAM in human health. Acknowledgments This was in part supported by the Multidisciplinary University Research Initiative (MURI) program of the Department of Defense (Biomimetic Cell and Tissue Stasis; N00014-01-1-0852), the Long Term Storage of Blood Products Program of DARPA (N00173-98-1-G005-P00004 and N0017302-1-G0 16), and a seed grant from the VT/UVA/Carilion Biomedical Institute Research Initiative (C.-S. L.). References Avalos, J.L., Bever, K.M., Wolberger, C., 2005. Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol. Cell 17, 855–868. Bitterman, K.J., Anderson, R.M., Cohen, H.Y., Latorre-Esteves, M., Sinclair, D.A., 2002. Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J. Biol. Chem. 277, 45099–45107. Blander, G., Guarente, L., 2004. The Sir2 family of protein deacetylases. Annu. Rev. Biochem. 73, 417–435. Chua, K.F., Mostoslavsky, R., Lombard, D.B., Pang, W.W., Saito, S., Franco, S., Kaushal, D., Cheng, H.L., Fischer, M.R., Stokes, N., Murphy, M.M., Appella, E., Alt, F.W., 2005. Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress. Cell Metab. 2, 67–76. Guarente, L., Kenyon, C., 2000. Genetic pathways that regulate ageing in model organisms. Nature 408, 255–262. Imai, S., Armstrong, C.M., Kaeberlein, M., Guarente, L., 2000. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800. Kaeberlein, M., Kirkland, K.T., Fields, S., Kennedy, B.K., 2004. Sir2-independent life span extension by calorie restriction in yeast. PLoS Biol. 2, e296. Krtolica, A., Parrinello, S., Lockett, S., Desprez, P., Campisi, J., 2001. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging. Proc. Natl. Acad. Sci. U.S.A. 98, 12072–12077. Langley, E., Pearson, M., Faretta, M., Bauer, U.M., Frye, R.A., Minucci, S., Pelicci, P.G., Kouzarides, T., 2002. Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. Embo J. 21, 2383– 2396. Luo, J., Nikolaev, A.Y., Imai, S., Chen, D., Su, F., Shiloh, A., Guarente, L., Gu, W., 2001. Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 107, 137–148. Matuoka, K., Chen, K.Y., Takenawa, T., 2001. Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation. Cell Mol. Life Sci. 58, 2108–2116.
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