In cancer therapy, chemotherapeutics with genotoxic activity are being used routinely. It is well known that these anticancer drugs induce DNA damage that triggers complex cellular DNA damage responses (DDR), which determine the fate of the cell, making the decision between survival and death [1
]. Key players involved in this scenario are the DDR kinases ATM, ATR and DNA-PK, the checkpoint kinases CHK1, CHK2, the stress kinase HIPK2, and further downstream, the transcription factor and tumor suppressor protein p53. During activation, p53 becomes phosphorylated, liberates from its inhibitor MDM1, becomes stabilized and binds as a transcription factor in a dimeric form to a p53 consensus sequence in the promoter of pro- and anti-apoptotic genes [2
]. In addition, p53 has other functions that are independent of transcriptional activation of genes [3
]. It is generally believed that low DNA damage levels activate pro-survival and high damage levels activate pro-death genes and cellular functions [4
]. For example, p53 stimulates the transcription of p21, which results in cell cycle arrest, and DNA repair genes such as DDB2, which enhances the repair capacity, leading to removal of toxic DNA lesions from DNA [7
]. At high dose levels p53 turns into a “killer” through activation of pro-death functions such as the proapoptotic genes Bax, Bak and Fas [2
]. Although this concept derived mostly from work with ionizing radiation is reasonable, there is not much experimental proof of it for chemical genotoxins, notably anticancer drugs. The concept implicates that there are threshold doses for cell death, i.e., low doses do not elicit activation of apoptosis pathways while high doses do.
A proof of this concept requires maximum understanding of the cell death pathways activated by a given genotoxicant. A well-studied drug in this respect is temozolomide (TMZ), which is used in first-line therapy for high-grade gliomas, including astrocytoma (WHO 0
3) and glioblastoma multiforme (glioma WHO 0
]. The main target of TMZ is the nuclear DNA in which, similar to other SN
1 alkylating agents, at least 12 nucleophilic sites can become methylated [9
]. The major methylation products are N
-methylpurines such as N7
-methylguanine and N3
-methyladenine, while O
-methylpurines are less frequent. Thus, O6
MeG) accounts for maximally 7% of the total methylations [9
]. Although produced in minor amounts, the damage is highly genotoxic and cytotoxic if not repaired by the suicide enzyme O6
-methylguanine-DNA methyltransferase (MGMT) [10
]. If cells are repair competent, O6
MeG is quickly removed from DNA. Under this condition, cells become highly resistant to O6
-alkylating agents and higher doses of a methylating agent are required to achieve a killing effect, which results from saturation of base excision repair and repair by ALKB homologous proteins (ALKBH) [11
]. Therefore, in the high dose setting, other lesions than O6
MeG, which are less toxic, give rise to cell death. The doses of TMZ in a therapeutic setting are very likely too low to achieve cell death resulting from non-repaired N-alkylations. Therefore, with an achievable serum concentration of up to 50 µM TMZ, the O6
MeG response plays a key role in determining tumor cell death.
The mechanisms of O6
MeG triggered genotoxic responses have been described previously [11
]. In brief, O6
MeG is a mutagenic mispairing lesion that results in mismatches with thymine that are subject to mismatch repair (MMR). Reinsertion of thymine during MMR causes a futile MMR cycle with gapped DNA that finally gives rise to DNA replication blockage and the formation of replication-mediated DNA double-strand breaks (DSBs), which occurs in the post-treatment cell cycle [12
]. These events provoke the activation of ATR und ATM, and downstream CHK1 and CHK2, respectively, as well as p53 phosphorylation [13
Upon genotoxic stress, p53 can be phosphorylated at different sites. p53 phosphorylated at serine 15 (p53ser15) and serine 20 (p53ser20) results from ATM/ATR-CHK2/CHK1 activation, while phosphorylation at serine 46 (p53ser46) results from activation of the kinase HIPK2 (for review, see [14
]). We have recently shown that this also occurs in glioblastoma cells upon treatment with TMZ. We also showed that p53ser46 exerts a pro-apoptotic function as downregulation of HIPK2, the kinase responsible for this phosphorylation, attenuated significantly the level of apoptosis in TMZ-treated LN-229 glioblastoma cells [15
In light of the hypothesis outlined above, according to which low doses elicit pro-survival and high doses pro-death functions, we wondered whether the dose-response of key players of DDR shows the hypothesized threshold. Here, we present data showing the non-existence of threshold doses for γH2AX, p53ser15, p53ser46, apoptosis, autophagy and senescence in the p53 expressing LN-229 glioblastoma cell system.
The DNA methylating agent temozolomide is a first-line drug in the treatment of high-grade malignant glioma. It is effective in inducing cell death if the tumor lacks MGMT or expresses it at low level, i.e., < 30 fmol/mg protein [22
]. These tumors are defined as “methylated” because of MGMT promoter CpG methylation, which correlates with silencing of the gene [23
] and deficient or low MGMT protein expression and enzyme activity [24
]. Since O6
MeG induced by TMZ (and other methylating anticancer drugs) is a toxic DNA damage, it is understandable that MGMT deficiency (determined, e.g., by promoter methylation) leads to responsiveness of the tumor [25
]. Despite these well-known relationships, the prognosis of glioblastoma, which account for up to 70% of high-grade malignant glioma, is bleak as the median length of survival is only 14.6 months (12.6 and 23.4 months in the MGMT-unmethylated and MGMT-methylated subgroups, respectively) [27
]. Although recent phase III clinical trials showed that the median overall survival for adult patients with newly diagnosed glioblastoma can reach up to 20 months in the control cohorts, indicating a trending increase in median overall survival, the prognosis is still bad with 5-year overall survival rates of less than 10% (for references see [28
]). Treatment with TMZ occurs daily along different schedules [29
]. The serum concentration of TMZ has been determined to be in the range of up to 30 µM, with a half-life of about 2 h [33
]. In a therapeutic setting with a single oral dose of 150 mg/m2
, the peak plasma concentration was, on average, 28.4 µM (5.5 µg/mL) and the brain interstitium concentration 1.5 µM (0.3 µg/mL) [39
]. In another study TMZ was determined following oral 200 mg/m2
TMZ, with a plasma peak level of 72 µM and a cerebrospinal fluid level of 9.9 µM [40
]. Thus, the TMZ concentration at the target organ seems to be rather low and it is reasonable to suppose, notably in view of the high recurrence rate, that the TMZ level is not high enough in order to exert a killing effect on residual (post-operative) glioblastoma cells. This notion is fueled by the supposition that at low dose levels cell death is not induced, which goes back to the general paradigm that low DNA damage levels induce survival functions, whereas high DNA damage levels activate cellular death pathways [1
]. This view implies that DNA damage thresholds do exist that regulate the balance between life and death. This work was aimed at proving or disproving this widely accepted hypothesis.
First, we have shown that in LN-229 and LN-308 glioblastoma cells, which are functionally wild-type and mutant for p53, respectively [16
], the amount of DSB (γH2AX foci) increases as a linear function of dose. TMZ does not need metabolic activation. It spontaneously decomposes, yielding carbenium ions that methylate DNA dose-dependently. From this it is reasonable to conclude that O6
MeG is induced as a linear function of dose. The linear dose-response for DSBs indicates that the rate of conversion of O6
MeG into DSB is independent on dose, and there is no defense at low dose levels that prevents the formation of DSBs in LN-229 and LN-308 cells. In this model system, we determined about 60 DSBs with a dose of 20 µM TMZ. The amount of O6
MeG induced under these conditions is not known.
We further show in Western blot experiments that p53, p-p53ser15 and p-p53ser46 increase up to a saturation level as a linear function of dose. This was a surprising finding since it collides with the view that low DNA damage triggers survival and high DNA damage triggers death functions. Upon TMZ treatment, p-p53ser15 results from ATR (ATM) and downstream CHK1 (CHK) activation [41
], which is likely the result of blocked replication forks and DSBs formed on collapsed forks. The linear dose-response suggests that even low O6
MeG and DSB levels induced by TMZ are able to activate the DNA damage checkpoint kinases that phosphorylate 53 at serine 15. Unexpectedly, p-p53ser46 was also generated at low dose levels without a detectable threshold. p-p53ser46 results from activation of the stress kinase HIPK2 [42
]. The pathway of HIPK2 activation in general [43
] and following TMZ in glioblastoma cells, including LN-229, has been described [15
]. The available data suggest that primarily ATR and following secondary activation, also ATM, phosphorylate SIAH1, the inhibitor of HIPK2 [44
]. This leads to degradation of SIAH1 and liberation and stabilization of HIPK2, which in turn phosphorylates p53 at serine 46 (see Figure 9
). The linearity of p-p53ser46 accumulation indicates that ATR (ATM) is able to phosphorylate SIAH1 and thus liberate HIPK2 even at very low TMZ doses.
We have shown that p-p53ser15 and p-p53ser46 become activated following TMZ treatment in a sequential order, with early activation of p-p53ser15 and late activation of p-p53ser46. p-p53ser15 becomes detectable 24 h after treatment and declines a day later, indicating that this is a transient response. p-p53ser46 was detected 3 days after treatment and was still detectable when cells started to undergo apoptosis. This is in line with the pro-apoptotic role of this phosphorylated form of p53.
In accordance with this is the finding that apoptosis (Figure 4
) and reproductive death (Figure 5
) of LN-229 cells do not display a clear no-effect threshold. Cell death appears to be a linear function of the dose of TMZ. It is known that p-p53ser46 binds to the promoter of pro-apoptotic genes, including the death receptor FAS (alias CD95/APO1), and thus stimulates its transcription [46
]. This was recently shown to occur upon TMZ in LN-229 cells [15
]. Obviously, there is no threshold for p-p53ser46 transactivation activity in this cell system.
Similar to apoptosis, DNA damage-induced senescence (Figure 6
) and autophagy (Figure 7
) were induced as a linear function of the dose of TMZ. In MGMT expressing cells, TMZ was ineffective in inducing these effects (doses up to 50 µM) suggesting that they were triggered by O6
MeG. Previously, we have shown that senescence and autophagy are regulated by the same upstream damage response pathway that regulates apoptosis [19
]. The linearity for these endpoints indicates that pro- and anti-death functions are induced simultaneously at each dose level. From the therapeutic point of view, the finding points to the need of inhibiting the pro-survival functions senescence and autophagy in a way that cells preferentially enter the death pathway.
Finally, we observed that the p53 deficient glioblastoma cell line, LN-308 is more resistant to the induction of apoptosis by TMZ. This finding is compatible with our previous observations according to which MGMT deficient p53wt glioma cells are more sensitive to the cytotoxic (apoptotic) effect of TMZ than MGMT deficient p53 mutated cells that lack the transactivation activity of p53 [47
]. The p-53 independent apoptotic pathway of glioblastoma cells is bound on the endogenous mitochondrial pathway, which seems to be more refractory than the p53 regulated death receptor pathway [18
]. Of note, p53 also stimulates the mitochondrial cell death route by supporting the translocation of BAX to the outer mitochondrial membrane and sequestering Bcl-2, leading to cytochrome C release and apoptosome formation [48
]. It is therefore reasonable to conclude that p53-driven apoptosis rests on both p-p53ser46 promoter activation and exacerbation of mitochondrial damage through cytoplasmatic p53.
4. Conclusions and Implications
Although we are aware that this study needs extension to other cell lines and tumor models, LN-229 provides an example where the paradigm that low doses activate survival and high doses death functions does not apply. Regarding DNA repair, it is known that p-p53ser15 triggers the activation of DNA repair genes, which causes protection against genotoxins [7
]. According to our experience with different cell types and genotoxins, the most robust p53-stimulated repair genes encode DDB2 and XPC as well as the translesion polymerase Pol eta (Pol H) [50
]. However, these genotoxic stress-inducible repair proteins are not involved in the repair of TMZ-induced DNA methylation damage. A reasonable candidate for causing a threshold is MGMT. Thus, from work that included bacteria to humans, it became clear that MGMT mediated DNA repair gives rise to a mutagenic and toxic threshold [11
]. However, a search for an adaptive response in brain cancer cells revealed that MGMT is not inducible by TMZ, which is clearly different from rodent cells in which MGMT was shown to be upregulated following genotoxic stress [53
] in a p53 dependent manner [54
]. Therefore, lack of induction of repair of O6
MeG in glioma cells is surely a contributing factor for the non-existence of a threshold. If ATR/ATM becomes activated even with low damage levels and also triggers senescence, autophagy and apoptosis, the important question arises as to the mechanism that makes the switch between the pathways. This is clearly an attractive area of future research.
In view of the limited amount of cell lines used in this study, it is too early for clinical implications. Nevertheless, the data may be taken to indicate that even a low dose of TMZ is able to elicit a cytotoxic response in p53 wild-type and MGMT lacking tumors. Of note, a prerequisite for O6MeG induced cytotoxicity is cell proliferation. If a fraction of tumor cells is released in a senescent state, it will no longer be subjected to O6MeG triggered apoptosis. This might especially be the case if cells are treated repetitively. Therefore, on the basis of the results presented here, the metronomic dose protocol (drug application at low and frequent doses) bears beneficial effects by exacerbating cytotoxicity, but also adverse effects since the fraction of non-proliferating (senescent) cells might be increasing with each consecutive treatment dose. It should also be considered that TMZ is usually given concomitantly with ionizing radiation (usually 2 Gy per treatment), which may additionally ameliorate the fraction of non-proliferating tumor cells. If the arrest state is transient, it is conceivable that the fraction of senescent cells at the end of therapy contributes to recurrence, which is usually the unfortunate case for glioblastomas. Again, we are aware of the limitations of the study, which rests on comparison of only three cell lines (LN-229, LN-220MGMT and LN-308). It provides, however, an example of lack of threshold doses in cell death responses (γH2AX, p53ser15 and p53ser46, apoptosis, autophagy and senescence) if MGMT is lacking. The data warrant further studies with a larger set of well-defined cell lines, stem cells and tumors in situ prior to and after therapy.
5. Materials and Methods
5.1. Cell Lines and Culture Conditions
The human glioma cell line LN-229 was purchased from American Type Culture Collection (ATCC), the human glioma line LN-308 were a generous gift from Prof. Dr. M. Weller (Laboratory of Molecular Neuro-oncology, University of Zurich, Switzerland). Upon receipt, the cells were amplified for cryopreservation in liquid-N2
and freshly thawed cell stocks were used for every battery of tests. LN-229, LN-308 and the LN-229MGMT transfected cells [19
] were cultured in DMEM (Gibco, Life Technologies Corporation, Paisley, UK) supplemented with 10% FBS and penicillin/streptomycin (PAA Laboratories, GmbH, Cölbe, Germany). Cells were maintained at 37°C in a humidified 5% CO2
5.2. Cell Seeding and Growth
Cells were cultured in DMEM supplemented with 10% fetal bovine serum. Cells were seeded 24 h before any treatment to settle down and get ready for knockdown and treatments. Seeding density was such that exponential cell growth was ensured for the whole experimental period.
5.3. Drugs and Drug Treatment
The MGMT inhibitor O6-benzylguanine (O6BG, Sigma-Aldrich, Steinheim, Germany) was dissolved in DMSO to a stock concentration of 10 mM, aliquoted and stored at −20 °C. To inactivate any residual MGMT, 1 h before the addition of TMZ O6BG was added to the medium. The final concentration of O6BG in DMEM was 10 μM. Temozolomide was a generous gift of Dr Geoff Margison (University of Manchester, UK). Stocks were dissolved in dimethyl sulfoxide (DMSO, Carl Roth GmbH, Karlsruhe, Germany), diluted in two parts sterile dH2O to a concentration of 35 mM, aliquoted and stored at −80 °C until use. After thawing, the stock solution was sonicated for 10 s to help TMZ dissolution. Cells were exposed to TMZ by directly adding the aqueous TMZ stock solution to the medium.
5.4. Colony Survival Assays
Cells were seeded in 6 cm dishes, treated 1 day later with TMZ and left to grow in a CO2 incubator until colonies appeared (microscopic control). Colonies were fixed in methanol and stained (1.25% Giemsa, 0.125% crystal violet). The plating efficiency represents the number of colonies formed in the control sample/ number of cells seeded in the control sample × 100%, and the surviving fraction is the number of colonies after treatment/number of cells seeded x PE. Colonies containing more than about 50 cells were scored.
5.5. Apoptosis/Necrosis Flow Cytometry
For the determination of apoptosis and necrosis the annexin V/propidium iodide (AV/PI) assay coupled with flow cytometry analysis was used. In brief, for harvest cells in the supernatant were collected in a 15 mL tube, samples were washed twice with PBS and detached with trypsin/EDTA solution. They were washed twice in PBS and 50 μL 1× binding buffer and 2.5 μL Annexin V/FITC (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added to each sample. Following 15 min incubation in the dark on ice, 430 μL 1x binding buffer and 1 µg/mL PI (Sigma-Aldrich, Steinheim, Germany) were added to the cells. Data acquisition was done by a FACS Canto II flow cytometer (Becton Dickinson GmbH, Heidelberg, Germany). Annexin V positive cells were classified as apoptotic while annexin V and PI double-positive cells were classified as necrotic/late-apoptotic. The data were analysed using the BD FACSDiva software. A representative plot of control and treated cells is shown in Supplementary Materials, Figure S1
5.6. Whole-Cell Protein Extracts
Cells were washed twice with PBS and 300-600ul RIPA buffer was added to each sample. The cells were scraped off and transferred to pre-cooled tubes, vortexed and put on ice. Sonication was employed for disrupting cells (3 × 10 pulses) and samples were centrifuged (10 min at 4 °C, 14,000 rpm) to obtain the protein extract in the supernatant. Protein concentration was determined by Bradford. The extraction buffer and RIPA buffer recipes were as follows: Extraction buffer: 20 mM tris(hydroxymethyl)aminomethane [TRIS] HCl pH 8.5, 1 mM EDTA, 5 % glycerine, 1 mM β-mercaptoethanol, 10 μM dithiothreitol [DTT], 1 x protease inhibitor cOmpleteTM. RIPA buffer: 50 mM Tris (pH 8), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS. This buffer was stored at 4 °C before use. Prior to use, freshly prepared PMSF 100 mM stock (10 μL), Na3VO4 200 mM stock (10 µL), DTT 1 M stock (2 μL) and 7x protease inhibitor (142.9 μL) were added to 835 µl RIPA buffer to get 1 mL working buffer.
5.7. Western Blot
Following the separation of proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to nitrocellulose membranes, the following antibodies were used: Anti-β-actin (Abcam; Ab8227), anti-HSP90 (Cell Signaling Technology, Frankfurt, Germany; No. 4874), anti-p53 (Santa Cruz Biotechnology, Heidelberg, Germany; sc-126), anti-phospho-p53 (Ser15) (Cell Signaling Technology; No. 9284), anti-phospho-p53 (Ser46) (Becton Dickinson; No. 558245), anti-MGMT (Sigma-Aldrich; HPA032136). Proteins were detected using the Odyssey 9120 Infrared Imaging System (Li-Cor Biosciences, Lincoln, Nebraska, USA). The membrane was dried at room temperature in the dark and scanned with Odyssey. Image J was used for the quantification.
5.8. Autophagy Assay
The Cyto-ID kit (ENZO Life Sciences, Lörrach, Germany) was used for quantifying autophagy. Cells were seeded in 6 cm dishes, being careful that cells were confluent when harvesting. The supernatant from each sample was transferred to a 15 mL tube, cells were rinsed with PBS and trypsinized with 1 mL trypsin-EDTA and taken up in 1 mL fresh medium, which was transferred to a 15 mL tube for centrifugation (1000 rpm, 5 min). The pellet was resuspended in 2 mL PBS, washed again in PBS and resuspended in 0.25 mL DMEM with 5% FBS without phenol red and 0.25 mL diluted Cyto-ID solution was added to each sample. After resuspension, the samples were incubated 30 min at 37 °C in the dark. After centrifugation (1500 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 mL assay buffer. Samples were centrifuged (1500 rpm, 5 min) and resuspended in 0.5 mL assay buffer and transferred into FACS tubes. FACS Canto was employed for the measurement. The data were analysed using the BD FACSDiva software.
5.9. The γH2AX Foci Assay
For measuring DSBs, the γH2AX foci assay was employed. The cells were seeded in 6 cm dishes in plates containing sterile cover slips. When harvesting, the medium was discarded, the samples were washed twice with PBS and cells were fixed in ice cold methanol:aceton (7:3 stored at −20 °C), kept on 4 °C for exactly 9 min. The fixation solution was removed, samples were rinsed three times with PBS and 2 mL PBS was added to each dish to keep the cover slips wet. The cover slip was put into a 3 cm dish (the cells side up), blocked with blocking buffer (5% BSA in PBS with 0.3% Triton X-100) for 1 h, the other cover slip was stored at 4 °C as a backup. The blocking buffer was removed, 50 µl of γH2AX antibody (Cell Signaling; Cat. No. 9718s) (1:1.000 dilution of γH2AX in PBS with 0.3% Triton X-100) was added on the cover slip for overnight incubation at 4 °C. After 3 times PBS washing, 50 µl of the secondary antibody (Alexa Fluor®
488, rabbit green, 1:500 of Alexa Fluor®
488 in PBS with 0.3% Triton X-100) was added to the cells on the cover slip and incubated at room temperature in the dark for 2 h, followed by three times washing with PBS. The secondary antibody (Alexa Fluor®
488) was from Life Technologies, Carlsbad, USA. DAPI-Vectashield (Vector Laboratories, Burlingame, CA, USA) and the solution (1.5 µl of 1 mg/mL DAPI was added in 1 mL Vectashield mounting medium, and vortexed thoroughly) was prepared freshly for staining. 20 µl of the DAPI-Vectashield solution was dropped on the center of one slide, the cover slip was put on the DAPI-Vectashield solution and sealed by nail oil. The slides were kept in the dark at room temperature for 10 min to dry. The γH2AX foci numbers were determined using the Metasystem finder version 3.1. Representative pictures of foci are shown in Supplementary Materials, Figure S2
5.10. Senescence Measurements with C12FDG Staining
Premature senescence was determined using C12
FDG and flow cytometry quantification. In brief, C12
FDG is a substrate of SA-β-galactosidase. Upon cleavage it produces a green fluorescence, which can be detected by FACS. Bafilomycin A1 is an inhibitor of vacuolar type H+
-ATPase (V-ATPase). It blocks lysosomal acidification and also increases the pH of lysosomes [55
]. Bafilomycin A1 (Sigma-Aldrich, Steinheim, Germany) was dissolved in DMSO at 0.1 mM stock solution and stored at −20 °C. The working concentration was 100 nM. C12
FDG (Sigma-Aldrich, Steinheim, Germany) was dissolved in DMSO (20 mM stock solution) and stored at -20 °C. The stock solution was diluted with fresh medium to get a 2 mM working solution. Cells were seeded and treated 96 h before the assay was performed. They were incubated with 100 nM bafilomycin A1 for 1 h and thereafter with 33 µM C12
FDG for 2 h. All the procedures after C12
FDG incubation were operated avoiding light. The samples were rinsed with PBS three times, 30s each, harvested with trypsin-EDTA and resuspended in serum containing medium together with the cells in the supernatant. They were collected by centrifugation at 4 °C, 100–250 g, 5 min. The pellet was resuspended in 0.4–0.5 mL PBS (4° C) and cells (titer of about 1 × 106
/mL) were measured in a FACS Canto II flow cytometer.
5.11. Statistical Analysis
If not clarified specifically, data points show the means of at least three independent experiments and the standard deviation from mean as error bars. For comparison, two-way ANOVA was employed, the calculated p-values are displayed: p-value < 0.05 *, p-value < 0.005 **, p-value < 0.001 ***, p-value < 0.0001 ****. GraphPad Prism software was used for statistical analysis and graph plotting.