We determined the effect of homocysteine treatment on cell survival in primary, differentiated cortical neurons. Cortical neurons isolated from embryonic day 18 embryos were placed in culture in Neurobasal medium for 7–10 days. On day 7, more than 90% of the cultured cells were positive for microtubule associated protein 2 staining (C), which is a marker specific for differentiated neurons (Mehra and Hendrickson, 1993
; Martinez et al., 1997
). Seven-day-old cultures were then treated with homocysteine at different doses and for the indicated times. The addition of homocysteine significantly reduced neuronal survival, in a dose- and time-dependent manner (A and B). Approximately 30% of the neurons died within 48
h in 0.25
mM homocysteine, and 80% died after 5 days of treatment. The 0.25
mM concentration of homocysteine was in the previously published range (Kruman et al., 2000
). As cell cycle activity is often density-dependent, we examined the response to homocysteine at different densities of cultured neurons, and reduced survival was seen in all conditions (A). Apoptosis activity in the homocysteine-treated neurons was detected by immunolabelling and confocal microscopy using anti-cleaved caspase-3 antibodies, suggesting that the neuronal death caused by homocysteine treatment was due to apoptotic activation (C and D). Less than 4% of the untreated cells were positive for anti-cleaved caspase-3 labelling and this low level presumably results from the low level of spontaneous death seen in our culture conditions. Homocysteine treatment increased cleaved caspase-3 detection to ~15% (C and D), which corresponded with the neuronal cell death seen in these populations (A and B).
Figure 1 Homocysteine induced apoptotic death in primary cortical neurons. (A) Cultured neurons, plated at the indicated densities were exposed to the indicated concentrations of homocysteine (Hcy) for 38h, before they were assessed for cell viability (more ...)
To determine whether homocysteine induces cell cycle re-activation in differentiated cortical neurons, we used BrdU to label newly synthesized DNA during the S phase of the cell cycle (Gonchoroff et al., 1986
). Few untreated neurons labelled with anti-BrdU antibodies, suggesting that the majority of the differentiated cortical neurons had exited the cell cycle and were not proliferating (C and D). A significant increase (>12%) of BrdU positive nuclei was found in the homocysteine-treated neurons (C and D), suggesting that these cells were actively synthesizing DNA. While in theory, DNA synthesis in response to DNA damage might increase the BrdU signal in the homocysteine treated cells, previous studies have shown that DNA repair does not result in significant BrdU labelling (Cooper-Kuhn and Kuhn, 2002
), suggesting that we were detecting DNA replication. Rb regulates cell cycle progression through the G1–S phase checkpoint (Sherr and Roberts, 1995
), and phosphorylation of Rb by cdks causes Rb inactivation and subsequent cell cycle progression (Knudsen and Wang, 1997
). We used anti-phospho-Rb (S795) immunolabelling to measure Rb phosphorylation in the presence or absence (control) of homocysteine treatment (C and D). Homocysteine treatment significantly increased the proportion of anti-phospho Rb (S795) positive nuclei from 18% in untreated cells to >40% in treated cells (C and D), suggesting that homocysteine treatment caused cell cycle reactivation and entry into S phase.
To correlate homocysteine-induced apoptosis with cell cycle phase, we stained cells with multiple antibodies simultaneously followed by confocal microscopy immunofluorescence. Co-localization of activated caspase-3 staining with BrdU labelling was seen in the nuclei of homocysteine-treated neurons (A). Cells were stained with anti-BrdU (blue), anti-phospho Rb (S795) (red) and TUNEL (green), and colocalization analysis of this triple labelling was performed in the homocysteine-treated neurons (B). The results show that colocalization between BrdU positive and TUNEL positive nuclei was almost 95% (B, right panel and 2C), suggesting that cells that initiated DNA synthesis and entered S phase were also undergoing apoptosis. Within the same group, less than 20% of cells contained both TUNEL and phospho-Rb (S795) staining. Likewise, <20% of the homocysteine-treated cells colocalized the BrdU and phospho-Rb (S795) markers (B, left, middle panels and 2C). While ~40% of the homocysteine-treated cells became Rb positive (C and D), the colocalization studies (B and C) suggest that Rb phosphorylation at the S795 site itself was not sufficient to induce apoptosis; and entry into S phase, as measured by BrdU incorporation and DNA synthesis, was required to cause neuronal cell death in differentiated cortical neurons. Homocysteine induced cell cycle re-entry in ~40% of the treated cells, but a smaller percentage (15–20%) entered S phase. This S phase entry, however, seemed sufficient to initiate apoptosis.
Figure 2 Multi-staining with BrdU, apoptosis and cell cycle markers in homocysteine-treated cortical neurons. Neurons were exposed for 24h to saline (control) or 0.25mM homocysteine (Hcy). BrdU was added to the medium to label DNA synthesis. (more ...)
To analyse cell cycle re-entry in the presence of homocysteine further, we examined many of the major cell cycle players that regulate the G1–S phase transition: cyclin D–cdk4, cyclin A–cdk2 and p27Kip1 (Sherr and Roberts, 1999
). Differentiated neurons were treated with homocysteine and analysed at different time points post treatment ( and ) to determine the expression levels, subcellular locations, interaction status and kinase activity of cdks and cyclin-dependent kinase inhibitors by immunoprecipitation, immunoblot and confocal microscopy. For microscopic analysis, co-staining with 4′,6-diamidino-2-phenylindole or ToPro3 was used to define nuclei (). We found that cyclin D1 immunoreactivity was detected mainly in the cytoplasm in differentiated cortical neurons (A and C). In homocysteine-treated neurons, increased nuclear and decreased cytoplasmic cyclin D1 was detected by 2
h, which continued to increase up to 20
h, suggesting that homocysteine treatment caused a cytoplasmic to nuclear translocation, similar to the mitogen-induced cytoplasmic to nuclear translocation of cyclin D1 observed in non-neuronal lineages (Gladden and Diehl, 2005
). Similar homocysteine-dependent translocations were seen with cdk4 and cdk2 (A). Cdk4 was localized in the cytoplasm of untreated neurons (0
h), but quickly accumulated in the nucleus in the presence of homocysteine (2
h), and was predominantly nuclear by 20
h of treatment (A). Cyclin A localization was nuclear in the untreated neurons, and was unaltered by homocysteine treatment. Cdk2 was predominantly cytoplasmic in untreated cells (0
h), but by 2
h of homocysteine treatment it was detected in the nucleus. Thus, in response to homocysteine treatment, cyclin D, cdk4 and cdk2 appeared to translocate to the nucleus, where they could potentially partner to form cyclin D–cdk4 and cyclin A–cdk2 complexes and phosphorylate their nuclear targets (Sherr and McCormick, 2002
). Rb phosphorylation itself was detected within 2
h of homocysteine treatment, suggesting that these neurons had re-entered the cell cycle and reached the G1–S phase border (A).
Figure 3 Immunoreactivity of G1 cell cycle proteins in cultured neurons. (A) Cultures were exposed for the indicated time courses to 0.25mM homocysteine (Hcy) before labelling with antibodies to cyclin D1, cdk4, cyclin A, cdk2, phospho-Rb (P-Rb) and p27Kip1. (more ...)
Figure 4 Expression and activities of cell cycle proteins in homocysteine-treated cortical neurons. Differentiated cortical neurons were pretreated with 4µM cdk2 inhibitor II 1h before adding homocysteine (Hcy) for the indicated times. (more ...)
p27Kip1 interacts with both cdk4 and cdk2-associated complexes, and is a potent inhibitor of these kinases in growth-arrested cells (Blain, 2008
; James et al., 2008
). It has been suggested that its high expression in differentiated cells may help to maintain cdk4 and cdk2 in their catalytically inactive forms, holding neuronal cells in the G0 quiescent state (Zindy et al., 1999
). To determine the localization and expression of p27, we stained homocysteine-treated neurons with anti-p27 antibodies. p27 was detected predominately in the nucleus of untreated differentiated neurons, with weaker but detectable cytoplasmic expression observed (A and B). In the presence of homocysteine by 20
h total p27 staining was reduced significantly (A). In apoptotic cells, the down-regulation of p27 appeared to occur significantly in the nucleus (B). As epithelial cells and lymphocytes exit G0 phase, p27 levels decrease to lower but detectable levels, restoring catalytic activity to inactive cdk complexes (Nickeleit et al., 2007
; Besson et al., 2008
), and these data in differentiated neurons were consistent with the idea that these cells had re-entered the cell cycle in response to homocysteine treatment.
Homocysteine-treated cells were analysed by immunofluorescence with multiple antibodies to determine co-localization of the different G1 cell cycle proteins (C). Double-staining with cyclin D1 and p27 antibodies demonstrated that cyclin D1 and p27 resided in different compartments in untreated cells: cyclin D1 was predominantly in the cytoplasm, while p27Kip1 was predominately in the nucleus (C). After homocysteine treatment, however, both cyclin D1 and p27 could be detected in the nuclear compartment in cells with intact nuclei as detected by ToPro nuclear staining (C). This nuclear to cytoplasmic translocation appeared to be a phenomenon specific for cyclin D1 and not cyclin D2 (D). Cyclin D2 was predominantly nuclear in untreated cells, and homocysteine treatment did not alter its localization (D).
While p27 levels appeared to remain high and nuclear in homocysteine-treated neurons that were not undergoing apoptosis, as measured by chromatin condensation (C), we predicted that p27 levels might decrease specifically in those neurons that were initiating apoptosis. Using anti-p27 antibodies, we found two types of p27 staining cells: homocysteine-treated cells that appeared to retain nuclear p27 (D, green) or alternatively those that had significantly reduced p27 expression (D, white dashed circles). Lack of nuclear p27 expression appeared to correlate with apoptosis, as measured visually by chromatin condensation seen in the ToPro stained cells (D, white dashed circles, merge). Homocysteine-treated cells with detectable p27 staining had intact nuclei, while cells with reduced or absent p27 staining were undergoing apoptosis. This suggested that p27 loss might be a prerequisite for apoptosis in differentiated neurons.
When homocysteine-treated and untreated neurons were stained with both anti-p27 and anti-phospho-Rb antibodies, an inverse correlation was observed (E). Little phospho-Rb was detected in untreated cells. By 7h post treatment phospho-Rb staining was seen, but only in cells that had severely reduced p27 expression (E, green arrows). In cells that retained high nuclear p27 expression, Rb phosphorylation was not detected, suggesting that reduction of p27 expression correlated with the appearance of phospho-Rb as a marker of the G1–S phase transition.
To demonstrate directly that neurons with activated G1 cell cycle programs were entering S phase, treated and untreated cells were co-stained with anti-cyclin D1 and BrdU antibodies (F). Little BrdU staining and predominantly cytoplasmic cyclin D1 were detected in untreated cells (0
h). Homocysteine treatment, however, caused the mobilization of cyclin D1. By 7
h of treatment, cyclin D1 was predominantly nuclear and BrdU specifically co-localized in these cells. Together these data suggest that homocysteine treatment caused the translocalization of cyclin D1, cdk4 and cdk2 to the nucleus, where they could potentially interact with their nuclear targets. Concomitant with this, a reduction to lower p27 levels was seen and Rb became phosphorylated. These data suggest that the differentiated neurons had exited the G0 phase, passing through G1 phase on their way to S phase. Co-localization of these events occurred in cells that underwent apoptosis, suggesting that the reactivation of the G1 cell cycle players and their activity was required for homocysteine-induced apoptosis.
Cyclin–cdks primarily phosphorylate nuclear targets during cell cycle progression. To examine the localization of the G1 cyclin–cdk complexes further, homocysteine-treated and untreated neurons were harvested at different times post homocysteine addition, and nuclear and cytoplasmic extracts were analysed by immunoblot analysis with antibodies to cyclin A, cyclin D1, cdk4 and cdk2 (A). Cyclin D1 was localized predominantly in cytoplasmic extracts in untreated neurons. Homocysteine treatment decreased its total levels, while significantly increasing its detection in the nucleus (A, lane 1). Cdk4 and cdk2 were also detected primarily in cytoplasmic extracts in untreated neurons, but by 4 and 9
h of treatment increased nuclear detection was observed (A, lanes 2 and 5). Cyclin A was detected in nuclear extracts in untreated neurons and the addition of homocysteine did not alter its distribution (A, lane 4), consistent with results seen by immunofluorescence microscopy. Cdk2 must be phosphorylated on a conserved threonine residue (T160) in order to be catalytically active (Sethi et al., 2005
). Using antibodies specific for cdk2 T160 phosphorylation, we did not detect active cdk2 in untreated cells, but it was detected in homocysteine-induced nuclear extracts within 4h of treatment, indicating that cdk2 had regained activity (A, lane 6), consistent with late G1 phase. Phosphorylated cdk2 was also detected in cytoplasmic extracts at 9
h of treatment, consistent with the increased activation and potential nuclear to cytoplasmic shuttling of this complex (A, lane 6). While the total level of Rb protein was unchanged by homocysteine treatment, nuclear (A, lane 8) phosphorylated Rb detection increased by 4
h of treatment (A, lane 7). These data were consistent with the results seen by confocal microscopy: the cyclins and cdks appeared to increase their nuclear expression and activation in the presence of homocysteine. We did not detect any cleaved caspase-3 expression in nuclear or cytoplasmic extracts in untreated neurons, but significant caspase-3 was detected in cytoplasmic extracts by 4
h of homocysteine treatment (A, lane 9), consistent with homocysteine’s ability to induce apoptosis following cell cycle re-entry.
Immunoprecipitation of treated and untreated lysates with cdk4 antibodies, followed by cyclin D1 immunoblot analysis, demonstrated that cyclin D1–cdk4 complex formation increased in the presence of homocysteine (C, lane 1). As the cdk4 monomer has minimal activity, increased complex formation should correspond to increased kinase activity (Bockstaele et al., 2006
). Immunoprecipitation with cdk4 antibodies, followed by the addition of recombinant Rb substrate and γ-ATP in an in vitro
kinase assay, demonstrated that homocysteine treatment increased cdk4 catalytic activity as well (C, lane 2).
Immunoblot analysis with antibodies specific for phosphorylated T160 cdk2 suggested that homocysteine-treatment increased cdk2 catalytic activity as well (A, lane 6). To demonstrate this directly, immunoprecipitation with cdk2 antibodies, followed by the addition of either recombinant Rb or Histone H1 substrates and γ-ATP in in vitro
kinase assays, demonstrated that cdk2 became catalytically active following homocysteine-treatment (C, lanes 4 and 5). Immunoblot analysis of total p27 levels demonstrated that p27 expression, which was high in untreated cells, decreased following homocysteine treatment (B), and this loss of p27 corresponded to a concomitant reduction of p27 in cdk2-associated complexes, as detected by cdk2 immunoprecipitation (C, lane 3). In untreated neurons, significant p27 was associated with cdk2, but this decreased to undetectable levels by 8
h of homocysteine treatment (C, lane 3). As p27 is a constitutive cdk2 inhibitor (Besson et al., 2008
), the lack of p27 in the cdk2 complex permits reactivation of catalytic activity, causing Rb phosphorylation and S phase progression. Our data suggest that homocysteine treatment rapidly reactivates cdk4 and cdk2 activity, permitting G1–S phase transitioning and Rb phosphorylation.
We postulated that if G1 cdk activation was required for apoptosis, inhibition of cdk activity might prevent homocysteine-induced neuronal death. We pre-treated neurons with several small molecule cdk inhibitors before the addition of homocysteine (A). Olomoucine is an inhibitor of cdks 1/2/5 and extracellular-signal-regulated kinase 1/microtubule associated protein kinase (Vesely et al., 1994
). It is a poor inhibitor of other protein kinases, and has been shown to prevent death due to nerve growth factor deprivation of differentiated PC12 cells and sympathetic neurons (Park et al., 1996
). K2 inhibitor II (Davis, et al., 2001
) and K4 inhibitor II (Kubo et al., 1999
) selectively inhibit cdk2 and cdk4, respectively. Pretreatment with olomoucine, K2 inhibitor II or K4 inhibitor II all protected neurons from homocysteine-induced death (A). Only 70% of cells remained viable after 48
h in the presence of 0.25
mM homocysteine. Treatment of cells with cdk inhibitors, however, increased cell survival (96.3%
K2 inhibitor II, P
K4 inhibitor II, P
0.01; or 92%
0.005) (A, left), suggesting that cdk activity was required for homocysteine-dependent apoptosis. K2 inhibitor II also rendered cells resistant to the neuronal death induced at higher concentrations of homocysteine (96.5% cells survival
K2 inhibitor II compared with 56.2% cells survival in 1
mM homocysteine alone) (A, right).
Immunoprecipitation with cdk4 antibodies, followed by cyclin D1 immunoblot analysis demonstrated that the homocysteine-dependent increase in cyclin D–cdk4 complex formation persisted in the presence of K2 Inhibitor II, when cdk2 was inhibited (C, lane 1), although catalytic activity of this complex was reduced (C, lane 2). Co-treatment with homocysteine and the K2 inhibitor II, however, prevented the homocysteine-dependent decrease in p27 levels (B), and immunoprecipitation of lysates with cdk2 antibodies, followed by p27 immunoblot analysis, demonstrated that p27 remained associated with cdk2 complexes (C, lane 3). p27 itself is a substrate of cdk2, and its phosphorylation by cdk2 increases its proteasomal degradation (Frescas and Pagano, 2008
). Immunoprecipitation with cdk2 antibodies, followed by the addition of either recombinant Rb or histone H1 substrates and γ-ATP in in vitro
kinase assays, confirmed the loss of cdk2 catalytic activity following homocysteine and K2 inhibitor II treatment. Thus, inhibition of cdk2 or cdk4 activity blocked cell cycle progression and correlated with increased cell survival in the presence of homocysteine treatment.
As an alternative to inhibit the G1 cdks, we attempted to reduce cyclin D1 expression by using antisense oligonucleotides (B). Sense and antisense oligonucleotides against cyclin D1 were transfected into differentiated neurons. Two days later cells were stained with anti-cyclin D1 antibodies and analysed by confocal immunofluorescence or harvested for immunoblot analysis with cyclin D1 antibodies (B, left). A significant reduction in cyclin D1 expression was detected by both methods and quantitated (B, left). After treatment with 0.25
mM homocysteine for 3 days, differentiated neurons transfected with antisense oligonucleotides showed significantly less apoptosis than cells that had been transfected with sense oligonucleotides (B, right). This was consistent with the idea that cyclin D1–cdk4 played an essential role in causing cell cycle re-entry and the concomitant neuronal apoptosis.
Other groups have suggested that the tyrosine kinase c-Abl becomes activated during the neuronal cell death induced by stressors such as β-amyloid fibrils (Alvarez et al., 2004
; Cancino et al., 2008
). To determine if Abl was involved in the regulation of homocysteine-dependent neuronal apoptosis, we examined untreated and homocysteine-treated cells by immunoblot analysis using Abl antibodies, and found that the expression of c-Abl increased with treatment time (C). We next treated neurons with Gleevec™
(STI571 mesylate salt), an inhibitor of c-Abl kinase activity (Avramis et al., 2003
; Hagerkvist et al., 2007
) before homocysteine treatment and measured cell survival. Treatment with Gleevec™
rescued neurons from homocysteine-induced cell death in a dose-dependent manner (D). Using immunofluorescent staining with multiple antibodies, we further evaluated the effects of Gleevec™
on G1 cell cycle activation (E). Gleevec™
treatment blocked the translocation of cyclin D1 and cdk4 from the cytoplasm to the nucleus in homocysteine-treated neurons (E), suggesting that c-Abl inhibition affected G1 phase progression. Gleevec™
treatment also reduced the detection of phospho-Rb after 7
h of homocysteine treatment (F), suggesting that these neurons had not progressed to nor activated the G1–S phase checkpoint. Thus, by chemical cdk inhibition of cdk2 and cdk4, decreasing cyclin D1 expression, or preventing cyclin D–cdk4 nuclear translocation via inhibition of c-Abl, we were able to block neuronal cell death, directly linking G1 cdk activation to homocysteine-induced apoptosis.
We wanted to determine whether blocking S phase progression was also sufficient to block homocysteine-induced death. We pretreated neurons with DNA replication inhibitors, such as aphidicolin, 1
mM hydroxyurea or 1
µM nocodazole, and 2
h later 0.25
mM homocysteine was added to the cultures as indicated (). Aphidicolin specifically inhibits DNA polymerase-α (Oguro et al., 1979
; Kowalzick et al., 1982
), hydroxyurea blocks ribonucleotide reductase (Adams and Lindsay, 1967
) and nocodazole inhibits microtubule dynamics and arrests the cell cycle at G2/M phase. Unlike the G1 phase inhibitors, none of these DNA replication inhibitors prevented neuronal death induced by homocysteine treatment (). In fact, treatment of neurons with nocodazole was toxic and co-treatment with homocysteine increased cell death even further. This suggests that once a cell entered S phase and began to replicate DNA, apoptosis was not preventable.
The DNA damage response pathway is activated by both physical DNA damage (DNA lesions and/or double- or single-stranded DNA breaks) or in response to DNA replication stress, due to stalled replication forks. The DNA damage response has been shown to be essential for the neuronal apoptosis due to DNA damaging agents, such as camptothecin (Rich et al., 2000
; Roos and Kaina, 2006
). We wanted to determine whether homocysteine treatment also caused DNA damage in cortical neurons. Using the Comet assay (Angelis et al., 1999
; Lemay and Wood, 1999
), which detects DNA single-strand breaks and incomplete excision repair sites, we showed that damage was detectable within 4
h of homocysteine treatment (A). More than 70% of the homocysteine-treated cells stained positively in this assay, whereas only a few Comet positive cells (<8%) were detected in non-treated neurons (A). Phosphorylation of H2AX (γ-H2AX) is an early sign of DNA damage induced by stalling replication forks (Bartek and Lukas, 2007
; Tanaka et al., 2007
; Chanoux et al., 2009
). By immunoblot analysis with γ-H2AX antibodies, we detected an increase in γ-H2AX following homocysteine treatment (B) suggesting that homocysteine-dependent DNA damage may be due to inappropriate DNA synthesis.
The central proteins of the DNA damage response are ATM and ATR (Rotman and Shiloh, 1999
; Pandita, 2002
; Brown and Baltimore, 2003
). While ATM and ATR share most downstream effectors, the ATM-Chk2 pathway is primarily activated by DNA double-strand breaks and the ATR-Chk1 pathway responds to stalled DNA replication forks (Shiloh, 2003
; Bartek and Lukas, 2007
). To determine the role of the DNA damage response pathway in homocysteine-induced apoptosis, we treated neurons with homocysteine and Ku-55933, an inhibitor of ATM protein (Hickson et al., 2004
) (A). We expected that if activation of the DNA damage response was responsible for triggering the cell death program, inhibition of ATM activity would block homocysteine-induced apoptosis. We found that Ku-55933 treatment did not rescue homocysteine-induced apoptosis, and in fact exacerbated it in a dose-dependent manner (A and B). Ku-55933 treatment in the absence of homocysteine had no affect on cell survival (A). Similar results were seen with caffeine treatment, which is a powerful inhibitor of ATR kinase (Sarkaria et al., 1999
; Lu et al., 2008
By immunoblot analysis with anti-cleaved caspase-3 antibodies using lysates from treated and untreated neurons, we saw that homocysteine-induced caspase-3 activation 8
h post treatment (E). Co-treatment with homocysteine and K2 inhibitor II prevented the detection of activated caspase-3, consistent with the reduced apoptosis seen in the presence of this inhibitor (A). Co-treatment with homocysteine and Ku-55933 or homocysteine and caffeine increased caspase-3 activation (E), consistent with the exacerbation of homocysteine-dependent apoptosis caused by inhibition of the ATM/ATR pathway seen in these cells (A–C).
Thus, inhibitors of ATR/ATM did not rescue homocysteine-induced neuronal cell death, suggesting that the DNA damage response pathway was not inducing apoptosis and might, in fact, normally function to prevent or slow homocysteine-induced apoptosis. This was in contrast to what was seen in camptothecin treated neurons (D). Camptothecin is a topoisomerase I inhibitor that induces apoptosis of cultured cortical neurons in a DNA damage dependent manner (Morris and Geller, 1996
; Staker et al., 2002
; Zhang et al., 2006
), and in our experiments, 10
µM camptothecin killed more than 40% of the treated neurons within 24
h (D). However, Ku-55933 treatment significantly enhanced cell survival levels, demonstrating that camptothecin-induced death was dependent on ATM activity and camptothecin-dependent activation of the DNA damage response pathway triggered apoptosis (D).
Activation of the ATR pathway was seen by immunoblot analysis using phospho-ATR antibodies that recognize the activated form of the protein, in both homocysteine and camptothecin-treated cells (A, lane 1; 9C, lane 1). ATM and ATR activation causes the phosphorylation and activation of Chk1 and Chk2 (Zhou and Elledge, 2000
; Brown and Baltimore, 2003
; Roos and Kaina, 2006
; Pabla et al., 2008
). Chk1 and Chk2 in turn can inhibit cell cycle progression, induce the expression of DNA repair enzymes, or ultimately mediate apoptosis if the damage is irreparable (Takai et al., 2000
; Maude and Enders, 2005
; Vitale et al., 2008
). Immunoblot analysis with phospho-Chk1 or phospho-Chk2 antibodies was performed with lysates from neurons treated with homocysteine. Both Chk1 and Chk2 were phosphorylated in response to homocysteine treatment (A, lanes 2 and 3, and 9B). Interestingly, we found that the two checkpoint kinases have different kinetic responses during the time course of homocysteine treatment. Detection of phospho-Chk2 increased rapidly between 1 and 4
h of exposure (A, lane 3), but decreased by 8
h. Phospho-Chk1 was not increased until 8
h and continued to increase up to 24
h of treatment (A, lane 2).
Figure 9 Homocysteine activates the ATR-Chk1/Chk2 pathway. Neurons were pretreated with Ku-55933, caffeine or K2 inhibitor II for 1h, before homocysteine (Hcy) addition for the indicated time. Cells were lysed and used in immunoblot analysis with (A) (more ...)
Treatment with both homocysteine and caffeine, which blocks ATR activation, reduced the detection of phospho-ATR (C, lane 1), while Ku-55933 treatment, which primarily inactivates ATM, did not affect ATR activation. Treatment of neurons with homocysteine and Ku-55933, however, significantly reduced but did not eliminate, Chk1 phosphorylation (B), verifying that Ku-55933 prevented the activation of the homocysteine-dependent DNA damage response pathway activation. Phospho-ATR, phospho-Chk1 and phospho-Chk2 were also detected following camptothecin treatment (A, lanes 1–3). When neurons were treated with both Ku-55933 and camptothecin, phospho-ATR and phospho-Chk1 detection was reduced, but phospho-Chk2 activity was blocked completely (A, lanes 1–3).
The tumour suppressor p53 has been shown to play an essential role in the apoptosis induction seen in response to DNA damage in camptothecin treated cortical neurons (Martin et al., 2008
). In the absence of DNA damage, p53 is poorly phosphorylated and rapidly degraded. In the presence of DNA damage, Chk1 and Chk2, among other proteins, can phosphorylate and stabilize p53. To determine whether p53 became phosphorylated and was stabilized in response to homocysteine-induced DNA damage, we used phospho-p53 antibodies in immunoblot analysis from lysates of treated neurons. Phosphorylation of p53 was not detected in untreated, differentiated neurons, but became weakly visible between 8 and 24
h of homocysteine treatment (A, lane 5). However, 8
h of campothecin treatment permitted the detection of strong p53 phorphorylation, which was lost in the presence of Ku-55933 treatment (A, lane 5). Thus, while DNA damage is seen in response to homocysteine treatment ( and C, lane 5), apoptosis induction by homocysteine seems p53-independent, and is distinct from the damage induced by campothecin.
Homocysteine treatment appeared to activate the DNA damage response pathway as seen by phosphorylation of ATR, Chk1 and Chk2, and Ku-55933 or caffeine treatment appeared to reduce this activation. Blocking the DNA damage response pathway by either Ku-55933 or caffeine, however, was unable to rescue neurons from homocysteine-induced death () and in fact exacerbated neuronal apoptosis. We wanted to determine whether blocking the DNA damage response pathway had any affect on cell cycle reactivation. Lysates from neurons treated with homocysteine and different inhibitors (Ku-55933, caffeine or K2 inhibitor II) were examined by immunoblot analysis to examine the levels of the G1 cell cycle proteins. Ku-55933 or caffeine pre-treatment did not block homocysteine-induced G1 cell cycle progression or activation: p27 levels were reduced (C, lane 2) and Rb became phosphorylated (C, lane 4), suggesting that homocysteine treatment initiated cell cycle re-entry independently of the activation status of the DNA damage response pathway. This was in contrast to what was seen when neurons were treated with homocysteine and K2 inhibitor II. As shown in B and C and A, and again in C, pre-treatment with K2 inhibitor II prevented homocysteine-induced cell cycle reactivation and blocked neuronal cell death. The homocysteine-dependent reduction in p27 levels was not detected (C, lane 2), and phospho-Rb was only weakly detected compared to the levels seen with homocysteine alone, homocysteine and Ku-55933 or homocysteine and caffeine (C, lane 4). Without cell cycle re-entry (no homocysteine or homocysteine and K2 inhibitor II treatment), H2AX phosphorylation was reduced, compared to levels seen in homocysteine alone or with homocysteine and Ku-55933 (C, lane 5). H2AX phosphorylation was detected even in the presence of Ku-55933 treatment (C, lane 5), as it can be phosphorylated by other replication stress-induced kinases, such as ATR or DNA-dependent protein kinase (Park et al., 2003
; Chanoux et al., 2009
). The homocysteine and K2 inhibitor II treated neurons only partially activated the DNA damage response pathway. Phospho-ATR was detected by immunoblot analysis (C, lane 1), but Chk1 phosphorylation was prevented by the presence of the K2 inhibitor II (B). While ATR was activated in response to homocysteine treatment, in the absence of cdk2 activity and cell cycle progression, it appeared unable to propagate the complete DNA damage response signal.