- Email: [email protected]
Autophosphorylation of H2AX in a cell-specific frozen dependent way
Cryobiology 57 (2008) 175–177
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Brief Communication
Autophosphorylation of H2AX in a cell-specific frozen dependent way q Lei Peng, Shouyu Wang, Shiwei Yin, Chunping Li, Zhong Li, Shoulin Wang, Qizhan Liu * Institute for Toxicology, School of Public Health, Nanjing Medical University, Nanjing 210029, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 17 November 2007 Accepted 19 June 2008 Available online 26 June 2008 Keywords: Frozen r-H2AX Autophosphorylation Protein lysate
a b s t r a c t Effective cryopreservation is a highly desired outcome in many laboratories including clinical and research centers. Cryopreservation-induced cellular damage through necrosis and apoptosis has been well characterized. However, our knowledge of the mechanism of induction of these cell death modalities is limited. H2AX histone phosphorylation to c-H2AX is a DNA damage response and is an excellent indicator of DNA double stranded break formation. In this study we examined and detected significant phosphorylation of H2AX in response to cryopreservation of HELF and B16 cell lines. The data provide strong evidence of intrinsic alterations in DNA repair pathway members for which the impact is yet to be fully understood. While further investigation will be necessary to characterize this response, our findings show a clear linkage between freezing resulting in phosphorylation and activation of the key DNA repair enzyme H2AX. Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved.
Effective cryopreservation and cold storage is becoming progressively more integral to day to day cellular biology as well as advances in cellular therapy [5]. The majority of cryopreservation protocols freeze cells in culture media with an additive such as dimethyl sulphoxide (DMSO) as the cryoprotective medium. Recent studies have shown that a large component of cryopreservation-related cellular damage is caused by delayed onset apoptosis which can be mitigated by inclusion of anti-apoptotic factors in cryopreservation media [1,2]. These results indicate that freezing does not just cause physiochemical stress and membrane disruption, which is largely mitigated by DMSO in cryopreservation, but that there are molecular-based damage mechanisms at play. As researchers become increasingly more dependent on the utilization of cryopreserved cells, characterization of the damage mechanisms associated with cryopreservation must continue to evolve. H2AX is one of the four histone proteins that make up the nucleosome unit of the chromatin [16]. The phosphorylation of H2AX is regarded as an important DNA damage signal and regulates a wide array of DNA damage response proteins. These proteins in turn act to repair the DNA damage caused by various stressors. H2AX is phosphorylated by the PI-3 kinase ataxia telangiectasia mutated (ATM) when it is in close proximity to a double stranded break in the DNA (DSB) [20,3]. Detection of the phosphorylated form of H2AX (c-H2AX) through immunoflourescence has been reported as an effective assay for DSB formation [22]. This method has been shown to detect DSB’s within minutes after
q
We do this research using the funding of National Natural Science Foundation of China (30571541), Natural Science Foundation of Jiangsu Province (BK2006233). * Corresponding author. Fax: +86 25 8652 7613. E-mail address: [email protected] (Q. Liu).
c-radiation exposure [19]. Another advantage of this technique is that it can reliably measure even small levels of DNA damage. For instance, Redon et al. [18] demonstrated that H2AX phosphorylation detection can measure the damage caused by doses of radiation as low as 1 mGy. Despite the relevance of H2AX as a cellular damage response mechanism, there are no studies in the literature characterizing the response of H2AX to freezing temperatures. In order to investigate the effect of freezing on intrinsic cell damage response mechanisms, we examined the relative levels of c-H2AX in human embryonic lung fibroblasts (HELF) and the B16 Mouse Melanoma cell line after exposure to freezing temperatures. H2AX phosphorylation has been detected in significant levels in cells undergoing apoptosis [8]. Therefore, to make sure that recorded H2AX phosphorylation was a cellular defense response to freezing temperatures versus a side effect of induced apoptosis or necrosis, various controls were included in our experiments. To control for catastrophic membrane failure, an additional set of samples were lysed prior to freezing. To account for apoptotic H2AX phosphorylation, samples were treated with 40 lM sodium arsenite [9]. Our results demonstrate that freezing cells using standard cryopreservation protocols induces H2AX phosphorylation beyond that caused by sodium arsenite exposure or cell lysis. H2AX phosphorylation increased dramatically in HELF and B16 lines after freezing at 20 and 80 °C The HELF and B16 cell lines were maintained according to standard protocols of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Both cell types were split into four test groups. Group One: the unfrozen control, Group Two: cells lysed and immediately frozen to 20 °C, Group Three: intact cells were
0011-2240/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.06.005
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L. Peng et al. / Cryobiology 57 (2008) 175–177
frozen to 20 °C, Group Four of intact cells frozen to 80 °C. After thawing, samples were analyzed for c-H2AX expression using a western blot procedure as described previously [11] (Fig. 1). The results demonstrate that freezing at either temperature yielded an increase in H2AX phosphorylation. This increase was observed regardless of whether cells were lysed or intact. These data indicated that the H2AX phosphorylation was in response to freezing as opposed to cellular rupture during the freezing process.
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H2AX phosphorylation in frozen and cold stored HELF unaltered by arsenite (NaAsO2) induced apoptosis In this study we compared H2AX phosphorylation in frozen cells which had been pre-treated with sodium arsenite and those which had not. This was done to determine the impact that freezing had on H2AX phosphorylation beyond induced apoptosis. All test samples of the HELF cell line were treated with 40 lM sodium arsenite (NaAsO2) for 24 h prior to freezing or cold storage. After washing with 0.9% sodium chloride, the cells were divided into six experimental groups. Two groups of HELF were stored at 4 °C for 1 h. One of these samples was exposed to NaAsO2, and both were lysed immediately prior to storage. Two groups of HELF were frozen at 20 °C with one group lysed prior to freezing. The final group was exposed to arsenite only. After treatment, all groups were then probed for c-H2AX presence as described previously. While the arsenite-exposed control showed some H2AX phosphyroyation, the frozen cells showed significantly higher levels of cH2AX (Fig. 2). The presence of c-H2AX was unaffected by arsenite pretreatment in frozen samples, indicating that the primary cause of H2AX phosphorylation in our experiment was the exposure of cells to low temperatures. Conclusions The above results indicate that the H2AX histone was phosphorylated in B16 and HELF cell lines in response to freezing. We found that cell lysis and induced apoptosis did not significantly alter the rates of H2AX phosphorylation in comparison to the damage caused during freezing. The phosphorylation of H2AX is a conserved damage response system, which indicates the formation of DSB. Our observations suggest two investigative angles into future cryopreservation work. Considering that H2AX phosphorylation was noted in cell lysates post thaw, it is possible that H2AX autophosphorylates in the presence of freezing temperature to preserve genetic integrity. It is also possible that freezing cells induces DSB formation through a mechanism such as oxidative stress. Cells exposed to low temperature conditions experience high degrees of oxidative stress [15]. There have also been studies
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r-H2AX β -Actin Fig. 1. H2AX phosphorylation increased dramatically in HELF and B16 lines after freezing at 20 and 80 °C. Both HELF and B16 cell types were split into four test groups. Group One: the unfrozen control, Group Two: cells lysed and immediately frozen to 20 °C, Group Three: intact cells were frozen to 20 °C, Group Four of intact cells frozen to 80 °C. The results demonstrate that freezing at either temperature yielded an increase in H2AX phosphorylation.
Fig. 2. H2AX phosphorylation in frozen and cold stored HELF unaltered by arsenite (NaAsO2) induced apoptosis. All test samples of the HELF cell line were treated with 40 lM sodium arsenite (NaAsO2) for 24 h prior to freezing or cold storage. Two groups of HELF were stored at 4 °C for 1 h. One of these samples was exposed to NaAsO2, and both were lysed immediately prior to storage. Two groups of HELF were frozen at 20 °C with one group lysed prior to freezing. The final group was exposed to arsenite only. While the arsenite-exposed control showed some H2AX phosphorylation, the frozen cells showed significantly higher levels of c-H2AX.
which demonstrate that both cold storage and cryopreservation significantly reduce cellular levels of glutathione [24,14]. This is a strong indicator that cells exposed to these conditions experience significant oxidative stress, as glutathione acts as the cell’s first line of defense against oxidative damage [4]. During cold storage of organs and cells, it has been shown that University of Wisconsin solution, which has strong antioxidant content, mitigates cell death and organ failure [23]. It is well known that oxidative damage can cause severe DNA damage, but the occurrence of this phenomenon in cryopreservation has not been well characterized. There have been studies published which demonstrate that cryopreserved sperm often show DNA damage on a comet assay [25]. However, these data have been primarily utilized to demonstrate the occurrence of apoptosis as opposed to heritable DNA damage. Our H2AX phosphorylation results indicate that there may be more long-term DNA integrity damage caused by the process of cryopreservation than previously thought. Cells are often put under conditions of intense heat and cold, and have evolved adaptive mechanisms to maintain cell viability and genetic integrity when exposed to such stressors. This capacity has been well characterized for heat in the production of heat shock proteins, which allow for proper protein folding under unfavorable energy conditions [17]. It has also been shown that biological systems have methods of adapting to extended exposure to cold temperature conditions. The wood frog Rana sylvatica has a signal phosphorylation cascade along the JNK pathway that is activated in response to extreme cold [6,7]. A recent liver transplantation study indicated that ground squirrels expressing the hibernation phenotype had livers which could survive cold storage much more effectively than rat livers cold-preserved in UW [12]. Hibernating animals have well understood defense mechanisms by which they can weather prolonged exposure to low temperatures. Currently, there has been very little work on the elucidation of the non-hibernating organism’s defensive response to freezing temperature. It has been shown that in response to freezing temperatures, myosin light chains in smooth muscle tissue become rapidly phosphorylated [13]. There has also been a study indicating that cryopreservation of porcine sperm initiated unknown protein phosphorylation signaling pathways [21]. These examples of protein phosphorylation lend credence to the possibility that H2AX autophosphorylation acts as a freezing defense mechanism. The results support this theory, as frozen cell lysates demonstrated similar levels of H2AX phosphorylation to unlysed samples. Characterization of this defense pathway would provide insight into the molecular mechanisms of low temperature-induced cellular damage.
L. Peng et al. / Cryobiology 57 (2008) 175–177
These data provide perspective into the molecular model of cryopreservation damage response by illustrating a DSB repair response to freezing temperatures. H2AX phosphorylation is an essential part of the cell’s DSB damage repair and response mechanism. DSB formation can result in severe and sometimes lethal mutations [10]. Our data show that freezing cells activates these defenses against DSB formation during the freezing process, which suggests that cells are exposed to DNA damaging factors as they are frozen. The survivors of cryopreservation may have a source of genetic variance and damage beyond our current understanding. In light of these results, it is clear that further analysis of the damage experienced by cells during freezing, and their defense against it is needed. Acknowledgments We thank Dr. John G. Baust for his precious advices and revision during the preparation of manuscript. This work was supported in part by National Natural Science Foundation of China (30571541), Natural Science Foundation of Jiangsu Province (BK2006233), Collegiate Natural Science Foundation of Jiangsu Province (04KJB330090). References [1] J.M. Baust, B. Van, J.G. Baust, Cell viability improves following inhibition of cryopreservation-induced apoptosis, In Vitro Cell Dev. Biol. Anim. 36 (2000) 262–270. [2] J.M. Baust, M.J. Vogel, R. Van Buskirk, J.G. Baust, A molecular basis of cryopreservation failure and its modulation to improve cell survival, Cell Transplant. 10 (2001) 561–571. [3] S. Burma, B.P. Chen, M. Murphy, A. Kurimasa, D.J. Chen, ATM phosphorylates histone H2AX in response to DNA double-strand breaks, J. Biol. Chem. 276 (2001) 42462–42467. [4] S. Chakravarthi, C.E. Jessop, N.J. Bulleid, The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress, EMBO Rep. 7 (2006) 271–275. [5] B. Fuller, N. Lane, E. Benson, Life in the Frozen State, CRC press LLC Forward by Meryman, 2004. [6] S.C. Greenway, K.B. Storey, Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog, Mol. Cell. Biochem. 209 (2000) 29–37. [7] C.P. Holden, K.B. Storey, Signal transduction, second messenger, and protein kinase responses during freezing exposures in wood frogs, Am. J. Physiol. 271 (1996) R1205–1211.
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[8] X. Huang, H.D. Halicka, F. Traganos, T. Tanaka, A. Kurose, Z. Darzynkiewicz, Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis, Cell Prolif. 38 (2005) 223–243. [9] V.N. Ivanov, T.K. Hei, Sodium arsenite accelerates TRAIL-mediated apoptosis in melanoma cells through upregulation of TRAIL-R1/R2 surface levels and downregulation of cFLIP expression, Exp. Cell Res. 312 (2006) 4120–4138. [10] J. Kobayashi, K. Iwabuchi, K. Miyagawa, E. Sonoda, K. Suzuki, M. Takata, H. Tauchi, Current topics in DNA double-strand break repair, J. Radiat. Res. (Tokyo) 49 (2008) 93–103. [11] H. Li, J. Wang, B. Kaphalia, G.A. Ansari, M.F. Khan, Quantitation of acroleinprotein adducts: potential biomarker of acrolein exposure, J. Toxicol. Environ. Health A67 (2004) 513–524. [12] S.L. Lindell, S.L. Klahn, T.M. Piazza, M.J. Mangino, J.R. Torrealba, J.H. Southard, H.V. Carey, Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype, Am. J. Physiol. Gastrointest. Liver Physiol. 288 (2005) G473–G480. [13] R. Maass-Moreno, T. Burdyga, R.W. Mitchell, C.Y. Seow, J. Ragozzino, L.E. Ford, Simple freezing apparatus for resolving rapid metabolic events associated with smooth muscle activation, J. Appl. Physiol. 90 (2001) 2453–2459. [14] J.F. McAnulty, X.Q. Huang, The efficacy of antioxidants administered during low temperature storage of warm ischemic kidney tissue slices, Cryobiology 34 (1997) 406–415. [15] M. Meseguer, N. Garrido, C. Simon, A. Pellicer, J. Remohi, Concentration of glutathione and expression of glutathione peroxidases 1 and 4 in fresh sperm provide a forecast of the outcome of cryopreservation of human spermatozoa, J. Androl. 25 (2004) 773–780. [16] J.D. Moore, J.E. Krebs, Histone modifications and DNA double-strand break repair, Biochem. Cell Biol. 82 (2004) 446–452. [17] L. Pirkkala, P. Nykanen, L. Sistonen, Roles of the heat shock transcription factors in regulation of the heat shock response and beyond, FASEB J. 15 (2001) 1118–1131. [18] C. Redon, D. Pilch, E. Rogakou, O. Sedelnikova, K. Newrock, W. Bonner, Histone H2A variants H2AX and H2AZ, Curr. Opin. Genet. Dev. 12 (2002) 162–169. [19] C. Redon, D.R. Pilch, E.P. Rogakou, A.H. Orr, N.F. Lowndes, W.M. Bonner, Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage, EMBO Rep. 4 (2003) 678–684. [20] E.P. Rogakou, D.R. Pilch, A.H. Orr, V.S. Ivanova, W.M. Bonner, DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139, J. Biol. Chem. 273 (1998) 5858–5868. [21] M.M. Satorre, E. Breininger, M.T. Beconi, N.B. Beorlegui, a-Tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm, Theriogenology 68 (2007) 958–965. [22] O.A. Sedelnikova, D.R. Pilch, C. Redon, W.M. Bonner, Histone H2AX in DNA damage and repair, Cancer Biol. Ther. 2 (2003) 233–235. [23] Y. Shirouzu, Y. Gu, M. Koga, T. Sakurai, M. Qi, A. Hiura, S. Sumi, K. Inoue, Cold preservation of islets in UW solution—with special reference to apoptosis, J. Surg. Res. 133 (2006) 167–175. [24] D.J. Stevenson, C. Morgan, L.I. McLellan, M. Helen Grant, Reduced glutathione levels and expression of the enzymes of glutathione synthesis in cryopreserved hepatocyte monolayer cultures,Toxicol. In Vitro 21 (2007) 527–532. [25] L. Zilli, R. Schiavone, V. Zonno, C. Storelli, S. Vilella, Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation, Cryobiology 47 (2003) 227–235.
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Brief Communication
Autophosphorylation of H2AX in a cell-specific frozen dependent way q Lei Peng, Shouyu Wang, Shiwei Yin, Chunping Li, Zhong Li, Shoulin Wang, Qizhan Liu * Institute for Toxicology, School of Public Health, Nanjing Medical University, Nanjing 210029, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 17 November 2007 Accepted 19 June 2008 Available online 26 June 2008 Keywords: Frozen r-H2AX Autophosphorylation Protein lysate
a b s t r a c t Effective cryopreservation is a highly desired outcome in many laboratories including clinical and research centers. Cryopreservation-induced cellular damage through necrosis and apoptosis has been well characterized. However, our knowledge of the mechanism of induction of these cell death modalities is limited. H2AX histone phosphorylation to c-H2AX is a DNA damage response and is an excellent indicator of DNA double stranded break formation. In this study we examined and detected significant phosphorylation of H2AX in response to cryopreservation of HELF and B16 cell lines. The data provide strong evidence of intrinsic alterations in DNA repair pathway members for which the impact is yet to be fully understood. While further investigation will be necessary to characterize this response, our findings show a clear linkage between freezing resulting in phosphorylation and activation of the key DNA repair enzyme H2AX. Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved.
Effective cryopreservation and cold storage is becoming progressively more integral to day to day cellular biology as well as advances in cellular therapy [5]. The majority of cryopreservation protocols freeze cells in culture media with an additive such as dimethyl sulphoxide (DMSO) as the cryoprotective medium. Recent studies have shown that a large component of cryopreservation-related cellular damage is caused by delayed onset apoptosis which can be mitigated by inclusion of anti-apoptotic factors in cryopreservation media [1,2]. These results indicate that freezing does not just cause physiochemical stress and membrane disruption, which is largely mitigated by DMSO in cryopreservation, but that there are molecular-based damage mechanisms at play. As researchers become increasingly more dependent on the utilization of cryopreserved cells, characterization of the damage mechanisms associated with cryopreservation must continue to evolve. H2AX is one of the four histone proteins that make up the nucleosome unit of the chromatin [16]. The phosphorylation of H2AX is regarded as an important DNA damage signal and regulates a wide array of DNA damage response proteins. These proteins in turn act to repair the DNA damage caused by various stressors. H2AX is phosphorylated by the PI-3 kinase ataxia telangiectasia mutated (ATM) when it is in close proximity to a double stranded break in the DNA (DSB) [20,3]. Detection of the phosphorylated form of H2AX (c-H2AX) through immunoflourescence has been reported as an effective assay for DSB formation [22]. This method has been shown to detect DSB’s within minutes after
q
We do this research using the funding of National Natural Science Foundation of China (30571541), Natural Science Foundation of Jiangsu Province (BK2006233). * Corresponding author. Fax: +86 25 8652 7613. E-mail address: [email protected] (Q. Liu).
c-radiation exposure [19]. Another advantage of this technique is that it can reliably measure even small levels of DNA damage. For instance, Redon et al. [18] demonstrated that H2AX phosphorylation detection can measure the damage caused by doses of radiation as low as 1 mGy. Despite the relevance of H2AX as a cellular damage response mechanism, there are no studies in the literature characterizing the response of H2AX to freezing temperatures. In order to investigate the effect of freezing on intrinsic cell damage response mechanisms, we examined the relative levels of c-H2AX in human embryonic lung fibroblasts (HELF) and the B16 Mouse Melanoma cell line after exposure to freezing temperatures. H2AX phosphorylation has been detected in significant levels in cells undergoing apoptosis [8]. Therefore, to make sure that recorded H2AX phosphorylation was a cellular defense response to freezing temperatures versus a side effect of induced apoptosis or necrosis, various controls were included in our experiments. To control for catastrophic membrane failure, an additional set of samples were lysed prior to freezing. To account for apoptotic H2AX phosphorylation, samples were treated with 40 lM sodium arsenite [9]. Our results demonstrate that freezing cells using standard cryopreservation protocols induces H2AX phosphorylation beyond that caused by sodium arsenite exposure or cell lysis. H2AX phosphorylation increased dramatically in HELF and B16 lines after freezing at 20 and 80 °C The HELF and B16 cell lines were maintained according to standard protocols of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Both cell types were split into four test groups. Group One: the unfrozen control, Group Two: cells lysed and immediately frozen to 20 °C, Group Three: intact cells were
0011-2240/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.06.005
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L. Peng et al. / Cryobiology 57 (2008) 175–177
frozen to 20 °C, Group Four of intact cells frozen to 80 °C. After thawing, samples were analyzed for c-H2AX expression using a western blot procedure as described previously [11] (Fig. 1). The results demonstrate that freezing at either temperature yielded an increase in H2AX phosphorylation. This increase was observed regardless of whether cells were lysed or intact. These data indicated that the H2AX phosphorylation was in response to freezing as opposed to cellular rupture during the freezing process.
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H2AX phosphorylation in frozen and cold stored HELF unaltered by arsenite (NaAsO2) induced apoptosis In this study we compared H2AX phosphorylation in frozen cells which had been pre-treated with sodium arsenite and those which had not. This was done to determine the impact that freezing had on H2AX phosphorylation beyond induced apoptosis. All test samples of the HELF cell line were treated with 40 lM sodium arsenite (NaAsO2) for 24 h prior to freezing or cold storage. After washing with 0.9% sodium chloride, the cells were divided into six experimental groups. Two groups of HELF were stored at 4 °C for 1 h. One of these samples was exposed to NaAsO2, and both were lysed immediately prior to storage. Two groups of HELF were frozen at 20 °C with one group lysed prior to freezing. The final group was exposed to arsenite only. After treatment, all groups were then probed for c-H2AX presence as described previously. While the arsenite-exposed control showed some H2AX phosphyroyation, the frozen cells showed significantly higher levels of cH2AX (Fig. 2). The presence of c-H2AX was unaffected by arsenite pretreatment in frozen samples, indicating that the primary cause of H2AX phosphorylation in our experiment was the exposure of cells to low temperatures. Conclusions The above results indicate that the H2AX histone was phosphorylated in B16 and HELF cell lines in response to freezing. We found that cell lysis and induced apoptosis did not significantly alter the rates of H2AX phosphorylation in comparison to the damage caused during freezing. The phosphorylation of H2AX is a conserved damage response system, which indicates the formation of DSB. Our observations suggest two investigative angles into future cryopreservation work. Considering that H2AX phosphorylation was noted in cell lysates post thaw, it is possible that H2AX autophosphorylates in the presence of freezing temperature to preserve genetic integrity. It is also possible that freezing cells induces DSB formation through a mechanism such as oxidative stress. Cells exposed to low temperature conditions experience high degrees of oxidative stress [15]. There have also been studies
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r-H2AX β -Actin Fig. 1. H2AX phosphorylation increased dramatically in HELF and B16 lines after freezing at 20 and 80 °C. Both HELF and B16 cell types were split into four test groups. Group One: the unfrozen control, Group Two: cells lysed and immediately frozen to 20 °C, Group Three: intact cells were frozen to 20 °C, Group Four of intact cells frozen to 80 °C. The results demonstrate that freezing at either temperature yielded an increase in H2AX phosphorylation.
Fig. 2. H2AX phosphorylation in frozen and cold stored HELF unaltered by arsenite (NaAsO2) induced apoptosis. All test samples of the HELF cell line were treated with 40 lM sodium arsenite (NaAsO2) for 24 h prior to freezing or cold storage. Two groups of HELF were stored at 4 °C for 1 h. One of these samples was exposed to NaAsO2, and both were lysed immediately prior to storage. Two groups of HELF were frozen at 20 °C with one group lysed prior to freezing. The final group was exposed to arsenite only. While the arsenite-exposed control showed some H2AX phosphorylation, the frozen cells showed significantly higher levels of c-H2AX.
which demonstrate that both cold storage and cryopreservation significantly reduce cellular levels of glutathione [24,14]. This is a strong indicator that cells exposed to these conditions experience significant oxidative stress, as glutathione acts as the cell’s first line of defense against oxidative damage [4]. During cold storage of organs and cells, it has been shown that University of Wisconsin solution, which has strong antioxidant content, mitigates cell death and organ failure [23]. It is well known that oxidative damage can cause severe DNA damage, but the occurrence of this phenomenon in cryopreservation has not been well characterized. There have been studies published which demonstrate that cryopreserved sperm often show DNA damage on a comet assay [25]. However, these data have been primarily utilized to demonstrate the occurrence of apoptosis as opposed to heritable DNA damage. Our H2AX phosphorylation results indicate that there may be more long-term DNA integrity damage caused by the process of cryopreservation than previously thought. Cells are often put under conditions of intense heat and cold, and have evolved adaptive mechanisms to maintain cell viability and genetic integrity when exposed to such stressors. This capacity has been well characterized for heat in the production of heat shock proteins, which allow for proper protein folding under unfavorable energy conditions [17]. It has also been shown that biological systems have methods of adapting to extended exposure to cold temperature conditions. The wood frog Rana sylvatica has a signal phosphorylation cascade along the JNK pathway that is activated in response to extreme cold [6,7]. A recent liver transplantation study indicated that ground squirrels expressing the hibernation phenotype had livers which could survive cold storage much more effectively than rat livers cold-preserved in UW [12]. Hibernating animals have well understood defense mechanisms by which they can weather prolonged exposure to low temperatures. Currently, there has been very little work on the elucidation of the non-hibernating organism’s defensive response to freezing temperature. It has been shown that in response to freezing temperatures, myosin light chains in smooth muscle tissue become rapidly phosphorylated [13]. There has also been a study indicating that cryopreservation of porcine sperm initiated unknown protein phosphorylation signaling pathways [21]. These examples of protein phosphorylation lend credence to the possibility that H2AX autophosphorylation acts as a freezing defense mechanism. The results support this theory, as frozen cell lysates demonstrated similar levels of H2AX phosphorylation to unlysed samples. Characterization of this defense pathway would provide insight into the molecular mechanisms of low temperature-induced cellular damage.
L. Peng et al. / Cryobiology 57 (2008) 175–177
These data provide perspective into the molecular model of cryopreservation damage response by illustrating a DSB repair response to freezing temperatures. H2AX phosphorylation is an essential part of the cell’s DSB damage repair and response mechanism. DSB formation can result in severe and sometimes lethal mutations [10]. Our data show that freezing cells activates these defenses against DSB formation during the freezing process, which suggests that cells are exposed to DNA damaging factors as they are frozen. The survivors of cryopreservation may have a source of genetic variance and damage beyond our current understanding. In light of these results, it is clear that further analysis of the damage experienced by cells during freezing, and their defense against it is needed. Acknowledgments We thank Dr. John G. Baust for his precious advices and revision during the preparation of manuscript. This work was supported in part by National Natural Science Foundation of China (30571541), Natural Science Foundation of Jiangsu Province (BK2006233), Collegiate Natural Science Foundation of Jiangsu Province (04KJB330090). References [1] J.M. Baust, B. Van, J.G. Baust, Cell viability improves following inhibition of cryopreservation-induced apoptosis, In Vitro Cell Dev. Biol. Anim. 36 (2000) 262–270. [2] J.M. Baust, M.J. Vogel, R. Van Buskirk, J.G. Baust, A molecular basis of cryopreservation failure and its modulation to improve cell survival, Cell Transplant. 10 (2001) 561–571. [3] S. Burma, B.P. Chen, M. Murphy, A. Kurimasa, D.J. Chen, ATM phosphorylates histone H2AX in response to DNA double-strand breaks, J. Biol. Chem. 276 (2001) 42462–42467. [4] S. Chakravarthi, C.E. Jessop, N.J. Bulleid, The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress, EMBO Rep. 7 (2006) 271–275. [5] B. Fuller, N. Lane, E. Benson, Life in the Frozen State, CRC press LLC Forward by Meryman, 2004. [6] S.C. Greenway, K.B. Storey, Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog, Mol. Cell. Biochem. 209 (2000) 29–37. [7] C.P. Holden, K.B. Storey, Signal transduction, second messenger, and protein kinase responses during freezing exposures in wood frogs, Am. J. Physiol. 271 (1996) R1205–1211.
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[8] X. Huang, H.D. Halicka, F. Traganos, T. Tanaka, A. Kurose, Z. Darzynkiewicz, Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis, Cell Prolif. 38 (2005) 223–243. [9] V.N. Ivanov, T.K. Hei, Sodium arsenite accelerates TRAIL-mediated apoptosis in melanoma cells through upregulation of TRAIL-R1/R2 surface levels and downregulation of cFLIP expression, Exp. Cell Res. 312 (2006) 4120–4138. [10] J. Kobayashi, K. Iwabuchi, K. Miyagawa, E. Sonoda, K. Suzuki, M. Takata, H. Tauchi, Current topics in DNA double-strand break repair, J. Radiat. Res. (Tokyo) 49 (2008) 93–103. [11] H. Li, J. Wang, B. Kaphalia, G.A. Ansari, M.F. Khan, Quantitation of acroleinprotein adducts: potential biomarker of acrolein exposure, J. Toxicol. Environ. Health A67 (2004) 513–524. [12] S.L. Lindell, S.L. Klahn, T.M. Piazza, M.J. Mangino, J.R. Torrealba, J.H. Southard, H.V. Carey, Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype, Am. J. Physiol. Gastrointest. Liver Physiol. 288 (2005) G473–G480. [13] R. Maass-Moreno, T. Burdyga, R.W. Mitchell, C.Y. Seow, J. Ragozzino, L.E. Ford, Simple freezing apparatus for resolving rapid metabolic events associated with smooth muscle activation, J. Appl. Physiol. 90 (2001) 2453–2459. [14] J.F. McAnulty, X.Q. Huang, The efficacy of antioxidants administered during low temperature storage of warm ischemic kidney tissue slices, Cryobiology 34 (1997) 406–415. [15] M. Meseguer, N. Garrido, C. Simon, A. Pellicer, J. Remohi, Concentration of glutathione and expression of glutathione peroxidases 1 and 4 in fresh sperm provide a forecast of the outcome of cryopreservation of human spermatozoa, J. Androl. 25 (2004) 773–780. [16] J.D. Moore, J.E. Krebs, Histone modifications and DNA double-strand break repair, Biochem. Cell Biol. 82 (2004) 446–452. [17] L. Pirkkala, P. Nykanen, L. Sistonen, Roles of the heat shock transcription factors in regulation of the heat shock response and beyond, FASEB J. 15 (2001) 1118–1131. [18] C. Redon, D. Pilch, E. Rogakou, O. Sedelnikova, K. Newrock, W. Bonner, Histone H2A variants H2AX and H2AZ, Curr. Opin. Genet. Dev. 12 (2002) 162–169. [19] C. Redon, D.R. Pilch, E.P. Rogakou, A.H. Orr, N.F. Lowndes, W.M. Bonner, Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage, EMBO Rep. 4 (2003) 678–684. [20] E.P. Rogakou, D.R. Pilch, A.H. Orr, V.S. Ivanova, W.M. Bonner, DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139, J. Biol. Chem. 273 (1998) 5858–5868. [21] M.M. Satorre, E. Breininger, M.T. Beconi, N.B. Beorlegui, a-Tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm, Theriogenology 68 (2007) 958–965. [22] O.A. Sedelnikova, D.R. Pilch, C. Redon, W.M. Bonner, Histone H2AX in DNA damage and repair, Cancer Biol. Ther. 2 (2003) 233–235. [23] Y. Shirouzu, Y. Gu, M. Koga, T. Sakurai, M. Qi, A. Hiura, S. Sumi, K. Inoue, Cold preservation of islets in UW solution—with special reference to apoptosis, J. Surg. Res. 133 (2006) 167–175. [24] D.J. Stevenson, C. Morgan, L.I. McLellan, M. Helen Grant, Reduced glutathione levels and expression of the enzymes of glutathione synthesis in cryopreserved hepatocyte monolayer cultures,Toxicol. In Vitro 21 (2007) 527–532. [25] L. Zilli, R. Schiavone, V. Zonno, C. Storelli, S. Vilella, Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation, Cryobiology 47 (2003) 227–235.