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DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines
Journal of Photochemistry and Photobiology B: Biology 77 (2004) 45–54 www.elsevier.com/locate/jphotobiol
DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines Shailja Singh, B.S. Dwarakanath *, T. Lazar Mathew Department of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Brig SK Mazumdar Road, Timarpur, Delhi 110054, India Received 2 December 2003; received in revised form 25 June 2004; accepted 12 August 2004 Available online 8 October 2004
Abstract The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1–20 lM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 lM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2–3 folds at 5 lM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G2 blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity. 2004 Elsevier B.V. All rights reserved. Keywords: Clonogenic survival; Apoptosis; Micronuclei; Cell cycle perturbation; PLDR; Hoechst-33342; Aphidicolin
1. Introduction Development of primary therapeutic agents and adjuvants as well as strategies that selectively eliminate neoplastic cells has been the major focus of research in experimental oncology over the last few decades. In radiation therapy, the induction and repair of DNA lesions are central among the various cellular responses [1] that determine cellular lethality. Both these processes are influenced by a number of physico-chemical as well as biological parameters such as chromatin structure [2], tissue organization, presence of antioxidants, pool sizes *
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1011-1344/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2004.08.009
of repair enzymes and nucleotides, cell cycle status of the cells as well as optimal energy supply. Therefore, adjuvants that differentially modify the induction and/or repair of DNA damage in tumor and normal cells can significantly improve the therapeutic efficacy. The DNA ligands bisbenzimidazole derivative Hoechst-33258 and its ethoxy substituted analogue Hoechst-33342 form strong and non-covalent linkage with adenine and thymine rich regions in the minor groove of DNA [3,4]. The DNA bound ligands alter the degree of chromatin condensation besides inhibiting the activity of topoisomerase enzymes involved in the different DNA transactions, viz. DNA replication, transcription, gene expression and DNA repair [5,6]. The ligands have been shown to reduce radiation induced single and
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double strand breaks in aqueous DNA solutions as well as in cells [7–9]. Administration of these ligands prior to ionizing radiation exposure has also been found to marginally reduce radiation induced cytogenetic damage and cell death in vitro under certain conditions [9,10], and to protect whole body irradiated mice by reducing hematopoietic damage and enhancing animal survival [11]. However, in contrast to pre-irradiation treatment, the post irradiation treatment of cells with these ligands has been reported to enhance cell death [9]. While reduction in the induction of DNA damage has been fairly well established to be due to free radical scavenging and DNA radical quenching, the exact mechanisms underlying the radiosensitization action of Hoechst33342 are not yet fully understood. An understanding of the varying responses observed among different cells to low LET radiation is confounded by the effects of the ligand on the induction as well as repair of DNA damage, besides other damage dependent processes. Therefore, it was considered worthwhile to investigate the effects of the ligand on the cellular response to UV damage, which is predominantly caused by molecular excitation process rather than by free radical mediated damage as observed in the case of low linear energy transfer (LET) radiation. Using established human tumor cell lines, we have initiated systematic studies to investigate the effects of the ligand on the molecular (DNA) and cellular responses to UV induced damage. Results of the studies on the cellular responses to UV induced damage in two human glioma cell lines (U-87 and BMG-1) are presented in this communication. Treatment induced cellular responses such as cell cycle perturbation, growth inhibition, potential lethal damage recovery (PLDR), clonogenic cell death, cytogenetic damage and apoptosis were investigated.
2. Material and methods 2.1. Tumor cell lines
nyl]-2,5 0 -bi-1H-benzimidazole) trihydrochloride), HankÕs balanced salt solution (HBSS), DulbeccoÕs modified phosphate buffered saline (PBS), DulbeccoÕs modified eagleÕs medium (DMEM), fetal calf serum (FCS), N[2-hydroxyethyl] piperazine-N 0 -[2-ethanesulfonic acid] (HEPES) buffer, propidium iodide (PI), 4,6-diamidino2-phenyl indole (DAPI), Ribonuclease-A (RNase-A), trihydro-chloride and trypsin were obtained from Sigma Chemical Co., USA. All other chemicals used in the present study were of analytical grade from BDH, Glaxo laboratories (Qualigens), SRL, and E-Merck, India. 2.3. Ultraviolet light irradiation and treatment procedure A germicidal lamp (emitting most of its power at 254 nm) was used as a source of UV irradiation. Cells were irradiated at a distance of 30 cm from the UV lamp on ice. The incident dose rate measured under these conditions with a radiometer was 5.15 J m2 s1 (Model No. IL 1400 A, USA). Cells were incubated with varying concentrations (1– 20 lM) of Hoechst-33342 in HBSS at 37 C for 1 h, both before and after UV irradiation (26.75 J/m2). Following irradiation cells were incubated for different time interval at 37 C in complete media for studying different cellular responses. 2.4. Clonogenic survival assay After harvesting with 0.05% trypsin, 150–4000 (depending on the treatment) cells were plated 8–10 h before treating with varying concentrations (1–20 lM) of Hoechst-33342 in DMEM at 37 C for 1 h before and after UV irradiation. After the treatment, cells were incubated in dark under humidified, 5% CO2 atmosphere at 37 C for 8–10 days to allow colony formation. Colonies were fixed with methanol and stained with 1% crystal violet. Colonies of more than 50 cells were counted and the plating efficiency (PE) and the surviving fraction (SF) were calculated as described earlier [12].
Human cerebral glioma cell lines BMG-1 and U-87 used in the present studies were grown as monolayer cultures in DMEM with 5% fetal calf serum for BMG-1 cells and DMEM containing 10% FCS for U-87 cells and antibiotics i.e. penicillin (100 units/ml), streptomycin (50 lg/ ml) and nystatin (2 lg/ml). Stock cultures were passaged every third day after harvesting the cells with 0.05% trypsin and seeding 8 · 103 cells/cm2 in tissue culture flasks to maintain the cells in the exponential phase. All experiments were carried out in exponentially growing cells.
2.5. Potentially lethal damage recovery
2.2. Chemicals
After treatment, cells were incubated in growth medium for varying intervals of time, harvested by trypsinization and counted in a hemocytometer (adherent + floating). Cell proliferation was calculated by computing
The bisbenzimidazole derivative Hoechst-33342 (bis benzimide (2 0 -[4-ethoxyphenyl]-5-[4-methyl-1-piperazi-
Cultures were grown to a confluence and treated with Hoechst-33342 and UV without fresh medium supply. After treatment, cells were incubated in HBSS containing 1% serum for varying time intervals, harvested and plated with growth medium for the assessment of colony forming ability as described above. 2.6. Cell proliferation kinetics
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the increase in cell number and the cell proliferation index P, calculated as P = Nt/N0, where, Nt is the number of cells at time t, N0, the number of cells at the time of treatment. 2.7. Cell cycle perturbations Flow cytometric measurements of cellular DNA content were performed with the ethanol (70%) fixed cells using the intercalating DNA fluorochrome, PI as described earlier [13]. Briefly, the cells (0.5–1 · 106) were washed in PBS after the removal of ethanol and treatment with ribonuclease-A (200 lg/ml) for 30 min at 37 C. Subsequently, cells were stained with PI (50 lg/ml) in PBS. Measurements were made with a laser based (488 nm) flow cytometer (Facs Calibur; Beckton-Dickenson, USA) and data acquired using the Cell Quest software (Beckton-Dickenson, USA). Cell cycle analysis was performed using the Modfit program (BecktonDickenson, USA). 2.8. Micronuclei formation Air-dried slides containing acetic acid–methanol (1:3 V/V) fixed cells were stained with 2-aminophenylindoledihydrochloride (DAPI) (10 lg/ml in citric acid (0.01 M), disodium phosphate (0.45 M) buffer containing 0.05% Tween-20 detergent) as described earlier. Slides were examined under the fluorescence microscope using an UV excitation filter. Fluorescent nuclei were visualized using a blue emission filter. Cells containing micronuclei were counted from >1000 cells by employing the criteria of Countrymen and Heddle [14]. The fraction of cells containing micronuclei, called the M-fraction (%) was calculated as follows: M fraction ð%Þ ¼ N m =N t 100; where Nm is the number of cells with micronuclei and Nt is the total number of cells analyzed. Since, micronuclei formation is linked to cell proliferation, the micronuclei frequencies were normalized with respect to the cell numbers. 2.9. Detection of apoptotic cells Morphologically, marked condensation and margination of chromatin, fragmentation of nuclei and cell shrinkage characterize apoptotic cells and a good correlation between these morphological changes and DNA fragmentation (ladder) as hallmarks has been demonstrated [15]. The percentage of cells undergoing apoptosis was determined microscopically and by flow cytometry using PI and DAPI labeled cells. At least 1000 cells were counted and the percentage of apoptotic cells determined from slides prepared as described under Section 2.8 for micronuclei formation.
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In flow cytometric DNA analysis, the presence of a hypodiploid (sub G0/G1) population (with PI stained cells, as described for cell cycle analysis) is indicative of an apoptotic cell population. Measurements of cellular DNA content were made from PI stained, ethanol fixed cells as described in Section 2.7. Analysis of the hypodiploid population was performed using the Modfit program (Beckton-Dickenson, USA).
3. Results 3.1. Clonogenic cell survival A dose dependent decrease in SF was found by macro-colony assay (clonogenecity) in both BMG-1 and U87 cell lines and the extent of cell death was marginally higher in BMG-1 as compared to U-87 cells (Fig. 1). An UV dose of 26.75 J/m2 was found to be the isosurvival dose for the therapeutically relevant gamma ray dose of 2 Gy. Effects of Hoechst-33342 on UV (26.75 J/m2) induced cell death were studied as a function of the Hoechst-33342 concentration (1–20 lM). Pre and post-irradiation treatments with Hoechst-33342 enhanced the UV induced cell death in BMG-1 cells, in a concentration dependent manner (Fig. 1), suggesting an inhibition in the repair processes and or enhanced damage fixation. This was in contrast to our earlier observations with gamma irradiation, where a small decrease in cell death was noted when Hoechst-33342 was added 1 h before irradiation [9]. The extent of cell death was marginally higher when cells were treated with Hoechst-33342 immediately following irradiation as compared to pretreatment with Hoechst-33342 at all the concentrations. However, at higher concentrations of Hoechst-33342 (20 lM) the cytotoxic effects of the combined treatment (Hoechst-33342+UV) were synergistic and the increase in cell death was 5 fold (SF = 0.03) higher than the expected value from an additive effect (SF = 0.15) in BMG-1 cells. Similar results were obtained with U-87 cells (Fig. 1). In both the cell lines investigated, the extent of sensitization was marginally reduced (10–15%) when Hoechst-33342 (1 and 5 lM) was added at various time intervals following UV irradiation, implying that Hoechst-33342 interferes with the repair processing of UV-induced lesions (Fig. 2). To examine whether the Hoechst-33342 induced modification of UV toxicity in these asynchronously growing cultures was related to the status of DNA synthesis, we treated cells with the DNA polymerase inhibitor aphidicolin for 15 min prior to and during a subsequent exposure to Hoechst-33342. In the presence of 10 lM aphidicolin, the fraction of BrdU labeled cells (indicative of actively DNA synthesizing cells) decreased from approximately 45% to less than 2% in 5 min (data
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Fig. 1. Effects of Hoechst-33342 (added 1 h before or after) on the survival of UV irradiated (26.75 J/m2) exponentially growing human glioma cell lines BMG-1 and U-87 studied by macro colony assay. Inset UV dose response survival of BMG-1 and U-87 cells. Data presented are mean values (±SD) from at least five independent observations.
not shown). Under these conditions, the sensitivity of unirradiated cells to Hoechst-33342 was reduced by nearly 2 fold, whereas a 3–4 fold reduction was observed in UV irradiated cells (Fig. 3), implying that the Hoechst-33342 induced enhancement of UV cytotoxicity was highly dependent on the ongoing DNA synthesis. Treatment of cells with aphidicolin alone produced minimal cell killing (SF = 0.8) (Fig. 3).
Fig. 2. Effects of varying the time between UV irradiation (26.75 J/m2) and addition of Hoechst-33342 on the survival of human glioma cell lines BMG-1 and U-87. Data presented are mean values (±SD) from at least three independent observations.
crease in the SF over time, implying PLDR. Survival of cells treated with UV and Hoechst-33342 was significantly reduced in a concentration dependent manner (Fig. 3). After 4 h post-treatment incubation survival was reduced 10 folds as compared to immediate plating. Longer incubation did not result in additional cell killing (Fig. 4). 3.3. Cell proliferation
3.2. Potentially lethal damage recovery To study the effects of Hoechst-33342 on PLDR, confluent arrested cells were incubated in suboptimal growth conditions (1% serum + HBSS) for different time intervals following UV and combined treatments (1 and 5 lM Hoechst-33342+UV). Cells held under these conditions following UV irradiation showed a steady in-
The effects of Hoechst-33342 on the proliferation of exponentially growing cells were studied following UV irradiation by monitoring the kinetics of cell growth. A concentration dependent reduction in the rate of cell proliferation was observed (Fig. 5), with a lag period followed by a recovery from growth inhibition at a concentration of 5 lM. However, a cytostatic effect up to 48 h,
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Fig. 3. Effects of DNA synthesis inhibitor aphidicolin on Hoechst33342 induced modification of UV cytotoxicity in exponentially growing BMG-1 cells. Aphidicolin (10 lM) added 5 min before exposure of cells to Hoechst-33342 (20 lM) and UV irradiation (26.75 J/m2) was present for 1 h along with Hoechst-33342. Values are mean (±SD) of three observations from three independent experiments.
followed by a marginal increase in the cell number in both the cell lines was observed at 20 lM Hoechst33342 (Fig. 5). 3.4. Cell cycle perturbations To investigate the role of cell cycle perturbations after UV irradiation due to the bound ligand, cell cycle distributions were analyzed by cytofluorimetric measurements of cellular DNA contents at various time intervals following the treatment. UV irradiation transiently delayed cell cycle progression, with a significant accumulation in G2–M phase after 48 h post-treatment incubation (Fig. 6). This delay was further enhanced by Hoechst-33342 (5 lM) in both cell lines. While the G2–M block was transient in BMG-1 cells, it was nearly irreversible in U-87 cells even up to 72 h (Fig. 6). At a higher concentration of Hoechst-33342 (20 lM), a profound decrease in G2–M phase cells was observed in both cell lines with a concomitant increase in cell death possibly due to apoptosis as indicated by the appearance of cell population with a hypodiploid DNA content (Fig. 6). 3.5. Cytogenetic damage Mitotic death (linked to cytogenetic damage) and interphase death (apoptosis) together account for the cytotoxicity of many physicochemical agents including
Fig. 4. Effects of Hoechst-33342 on PLDR in BMG-1 and U-87 cell line studied by macro colony assay. Values are mean (±SD) of three observations from three independent experiments.
UV, although the relative contributions of the two death processes vary among the type of damaging agent. To investigate the modifications of UV-induced cytogenetic damage by Hoechst-33342, we studied the treatment-induced micronuclei formation in both cell lines. In UV irradiated cells, strand breaks generated during the excision repair process remaining unsealed lead to the formation of micronuclei in subsequent mitosis, and cells with micronuclei are found to be associated with loss of reproductive capacity [17]. Since cell proliferation influences treatment induced micronuclei expression, the data from kinetic studies up to 72 h post treatment were analyzed. The frequency of unirradiated cells with micronuclei was in the range of 1–2%, and treatment with 5 lM Hoechst-33342 did not induce a significant level of micronuclei formation in these cells (63%).
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sub G0/G1 population showed that UV and Hoechst33342 alone (5 and 20 lM) did not induce a significant level of apoptosis in both cell lines. However, the combined treatment significantly enhanced apoptosis in a time and concentration dependent manner in both the cell lines (Fig. 6). The frequency of apoptotic cells increased by nearly 50% following treatment with 20 lM Hoechst-33342 and UV at 72 h. The non-apoptotic population consisted of predominantly G1 and S and to a lesser extent of cells in G2–M phase cells (Fig. 6). Further, these results were supported by the morphological analysis of apoptosis (Fig. 8).
4. Discussion
Fig. 5. Effects of Hoechst-33342 (5 and 20 lM) on the proliferation of exponentially growing BMG-1 and U-87 cells. Cells were exposed to Hoechst-33342 for 1 h in HBSS immediately after UV irradiation. Data presented are mean values (±SD) from at least four independent observations. Where not shown, the SDÕs were less than the size of the symbols.
However, micronuclei frequency increased from 5% in UV irradiated cells to 12% with the combined treatment (5 lM Hoechst-33342+UV) at 48 h in BMG-1 cells following a decrease at 72 h. A comparable increase in U87 cells was also observed at 48 h, which however increased further at 72 h (Fig. 7). Due to extensive damage (interphase death), micronuclei expression could not be analyzed in cells treated with a higher concentration of Hoechst-33342 (20 lM) following UV irradiation. 3.6. Apoptosis The apoptotic mode of cell death in these cell lines was confirmed by microscopy and flow cytometry: the changes in cell morphology were characterized by typical chromatin condensation and nuclear fragmentation, with disruption of plasma membrane integrity and the appearance of a cell population with a hypodiploid DNA content. Flow-cytometric analysis of hypodiploid
Results of the present studies clearly demonstrate that both pre and post UV irradiation treatments with the DNA ligand Hoechst-33342 sensitizes cells in a concentration dependent manner, by increasing both the UV induced mitotic (linked to cytogenetic damage) and interphase (apoptosis) death. This is in contrast to ionizing radiation, where pre-irradiation Hoechst33342 treatment has been found to moderately decrease the lethality under certain condition [9]. Since UV irradiation causes mainly pyrimidine dimers due to direct UV absorption by DNA unlike the induction of DNA damage by low LET ionizing radiation via the generation of free radicals, the effects of Hoechst33342 on UV irradiated cells may be mainly due to its interference with repair and with the resolution of cleavable complexes. On the other hand, a net effect consequent to the alterations (mainly a reduction) in the damage induction as well as inhibition of repair processes determines the modification of cellular responses to ionizing radiation damage by Hoechst33342. In this respect, therefore studies on the effects of Hoechst-33342 on cellular responses to UV damage could be useful in elucidating its role in the biological response to radio-modification, besides the known effects of the ligand on radio-chemical aspects of damage induction [18]. In both the cell lines investigated here, the extent of sensitization was marginally reduced (10–15%) when Hoechst-33342 (5 lM) was added at various time intervals following UV irradiation (Fig. 2), implying that lesions whose processing is interfered by Hoechst-33342 are repaired to a certain extent with time. Hoechst33342 exhibits its cytotoxicity by interfering with the breakage-reunion reaction of DNA topoisomerases leading to the accumulation of intermediates termed Ôcleavable complexÕ [5,6]. At higher concentrations, the cytotoxic effects of the combination of Hoechst-33342 and UV were highly synergistic suggesting the possible interaction of lesions caused by both the agents i.e. UV and Hoechst-33342.
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Fig. 6. Flow cytometric DNA histograms from a typical experiment showing the effects of Hoechst-33342 on UV induced G2 block and apoptosis (arrow) in exponentially growing BMG-1 and U-87 cells.
Interestingly, under liquid holding conditions (HBSS + 1% serum), the DNA bound ligand not only inhibited the recovery from potentially lethal damage but also resulted in a time dependent increase in cell killing (Fig. 4). Fixation of many potentially lethal damage including part of the thymine dimers induced by UV irradiation under several conditions including those, which favour chromosome condensation [19] is expressed as enhanced chromosomal aberrations manifesting in mitotic death [20,21]. It appears therefore, that alterations in chromatin dynamics induced by the bound ligand [3,4] not only interferes in the repair, but also facilitates lesion fixation. Moreover, Hoechst-33342 remains bound to the cellular DNA for long intervals of time and stabilizes cleavable complex mediated DNA strand breaks, whose interaction with the primary UV lesion can also enhance lethality under these conditions. UV induced DNA lesions viz. thymine dimers and 6-4 photoproducts, block the DNA replication [22],
leading to the formation of DNA double strand breaks and chromosomal aberrations giving rise to acentric fragments because of nuclease attack at stalled replication forks [23,24]. When strand breaks generated during the repair of these lesions remain unligated, it results in chromosomal aberrations [14], which manifest as micronuclei in the subsequent mitosis. Micronuclei formation is therefore, one of the good indices to measure the residual DNA damage and loss of reproductive ability of cells [17]. The enhanced cell death due to Hoechst-33342 was also accompanied by a significant increase (2–3 folds at 5 lM Hoechst-33342) in UV induced micronuclei formation in both the cell lines (Fig. 7). This result suggests that the bound ligand profoundly inhibits the rejoining of strand breaks created during the excision repair (the major pathway for the repair of UV lesions), although it could interferes with the initial events of damage recognition and strand scission as
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Fig. 7. Effects of Hoechst-33342 (5 lM) on the kinetics of micronuclei expression following UV irradiation in exponentially growing BMG-1 and U-87 cells plotted as a function of time. Values are mean (±SD) of three independent observations.
well. Indeed inhibition of excision repair following UV induced DNA damage by the bound ligand has been observed (Shailja Singh et al., unpublished observations). However, the ligand may also induce a higher rate of faulty mitosis in damaged cells, which could partly contribute to the enhanced micronuclei observed. One of the characteristics of cellular response to UV irradiation is the accumulation of cells in S as well as G2–M phases of the cell cycle due to the inhibition of the replication by UV lesion, which was indeed observed in both the cell lines investigated (Fig. 6). In case of Hoechst-33342 bound cells, the transient accumulation of cells in G2–M phases of cell cycle is likely to be due to the presence of low levels of cleavable complexes (protein linked DNA breaks) as well as interference in chromosome segregation on account of topoisomerase II inhibition [25]. Cell cycle delay arises as an account of the operational functional checkpoints at G1–S and G2–M transition and the ex-
Fig. 8. Effect of Hoechst-33342 (20 lM) on UV induced apoptosis studied as a function of post-irradiation time in BMG-1 and U-87 cells. Data presented are mean values (±SD) from at least three independent observations.
tent of delay is related to the level of DNA damage [26]. The enhanced delay observed in Hoechst-33342 bound cells, therefore, indicates the presence of a higher level of DNA damage following UV irradiation. Since the cytotoxic effects of Hoechst-33342 has been found to be significantly higher in the S phase cells than in the G1 and G2–M phases of cell cycle [27], prolonged S phase by UV irradiation seems to potentiate the cytotoxic effects of the combined treatment (Hoechst-33342+UV) by inducing a larger cell population to be in the sensitive state for Hoechst33342 induced cell killing. The marked S phase specificity is thought to result from the interaction of topo I cleavable complex with replication fork resulting in the generation of lethal DNA double strand breaks like in case of the well known topoisomerase I inhibitor camptothecin [16,28]. Consistent with this hypothesis, we have observed a reduction in the Hoechst-33342 induced UV sensitization with DNA a polymerase inhibitor aphidicolin in BMG-1 cells (Fig. 3). In fact, the induction of double strand break in replicating DNA
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due the collision of replication fork with topoisomerase I involved cleavable complex has been proposed as a cause for the S phase specific lethal effects of topoisomerase I inhibitors [29]. Loss of clonogenicity following the induction of DNA damage could arise either due to the mitotic death and/or due to apoptosis depending on the cell type. Since the principle mechanisms of apoptosis differ in several ways from that of micronuclei formation, these are independent predictors of cellular sensitivity [30]. Nearly 50% increase in the UV induced apoptosis observed in both the cell lines at higher concentrations of Hoechst-33342 (20 lM) imply that the enhancement in cell death is significantly mediated through enhanced apoptosis as well (Fig. 6). Further, it was also found that non-apoptotic population consisted of predominantly G1 and S phase cells to a lesser extent G2–M phase cells. Programmed cell death (apoptosis) induced by UV-C light in various cell types has been proposed to be a consequence of receptor activation [31], as well as by p53 activation due to the UV induced DNA lesions stalling the transcription machinery [32,33]. Recently it has been also proposed that double strand breaks are the ultimate trigger of UV-C induced apoptosis [34]. Lesions induced by UV can be reversed by a number of repair pathways, which involve several gene products viz. endonucleases or exonucleases, polymerases and ligases [35,36]. Important roles for topoisomerase can be envisioned in these repair steps, and circumstantial evidences currently available indicate the participation of topoisomerase I and II in the repair of various forms of the DNA damage [36–38]. Contrary to its role in the repair of DNA damage, topoisomerases have been implicated in the generation of potentially lethal DNA breaks by interaction with UV induced DNA damage as well as with topoisomerase specific poisons [39,40]. Further, topoisomerase cleavable complex mediated breaks have been shown to induce apoptosis [41,42]. Therefore, it appears that inhibition in the repair of UV induced lesions as well as formation cleavable complex mediated, S phase dependent and/or independent strand breaks are to a very great extent responsible for the enhanced apoptosis in cells following the combined treatment with Hoechst33342 and UV-C.
Acknowledgements We thank Maj. Gen. T. Ravindranath Director, INMAS for his constant support and encouragement. Useful discussion during the initial part of this work with Prof. Viney Jain, Wright State University, USA is gratefully acknowledged. We thank J.S. Adhikari, Dr Seema
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Gupta and Rohit Mathur for helpful discussions and technical support.
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[18] A. Adhikari, E. Bothe, V. Jain, C. von Sonntag, Inhibition or radiation-induced DNA strand breaks by Hoechst-33258: OHradical scavenging and DNA radical quenching, Radioprotection 32 (1997) C1–C89. [19] G. Iliakis, Characterization and properties of repair of potentially lethal damage as measured with the help of b-arabinofuranosyladenine in plateau-phase EAT cells, Radiation Res. 86 (1981) 77– 90. [20] R.A. Phillips, L.J. Tolmach, Repair of potentially lethal damage in X-irradiated HeLa cells, Radiation Res. 29 (1966) 413–432. [21] B.K. Thomas, J.K. Levinson, V.M. Maher, J.J. McCormik, Correlation among the rates of dimer excision DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation, BioPhys. J. 28 (1979) 315–326. [22] R.B. Painter, R. Howard, The HeLa DNA synthesis test as a rapid screen for mutagenic carcinogens, Mutation Res. 92 (1982) 427–437. [23] B. Kaina, The interrelationship between SCE induction cell survival, mutagenesis, aberration formation and DNA synthesis inhibition in V79 cells treated with N-methyl-N-nitrosourea or Nmethyl-N 0 -nitro-N-nitrosoguanidine, Mutation Res. 142 (1985) 49–54. [24] B. Kaina, Critical steps in alkylation-induced aberration formation, Mutation Res. 404 (1998) 119–124. [25] T. Uemura, H. Ohkura, Y. Adachi, K. Morino, K. Shiozaki, M. Yanagida, DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe, Cell 50 (1987) 917–925. [26] A. Maity, W.G. Mckenna, R.J. Muscchel, The molecular basis for cell cycle delays following ionising radiation review, Radiother. Oncol. 31 (1994) 1–13. [27] W.S. Dietmar, C.K. Peter, Cell cycle specific toxicity of the Hoechst-33342 stain in untreated or irradiated murine tumor cells, Cancer Res. 46 (1986) 3556–3559. [28] K.-C. Chow, W.E. Ross, Topoisomerase-specific drug sensitivity in relation to cell cycle progression, Mol. Cell Biol. 7 (1987) 3119– 3123. [29] C. Holm, J.M. Covey, D. Kerrigan, Y. Pommier, Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells, Cancer Res. 49 (1989) 6365–6368.
[30] G.Z. Guo, K. Sasai, N. Oya, T. Takagi, K. Shibuya, M. Hiraoka, Simultaneous evaluation of radiation induced apoptosis and micronuclei in five cell lines, Int. J. Radiat. Biol. 73 (1998) 297– 302. [31] T. Schwarz, UV light affects cell membrane and cytoplasmic targets, J. Photochem. Photobiol. 44 (1998) 91–96. [32] M. Ljungman, F. Zhang, Blockage of RNA polymerase as a possible trigger for UV light-induced apoptosis, Oncogene 13 (1996) 823–831. [33] M. Ljungman, F. Zhang, F. Chen, A.J. Rainbow, B.C. Mckay, Inhibition of RNA polymerase II as a trigger for the p53 respons, Oncogene 18 (1999) 583–592. [34] R.D. Torsten, B. Kaina, Cell proliferation and DNA breaks are involved in ultraviolet light induced apoptosis in nucleotide excision repair-deficient Chinese Hamster cells, Mol. Biol. Cell 12 (2002) 348–361. [35] J.H.J. Hoeijmakers, D. Bootsma, Molecular genetics of eukaryotic DNA excision repair, Cancer Cell 2 (1990) 311–320. [36] K.B. Tan, M.R. Mattern, R.A. Boyce, P.S. Schin, Elevated topoisomerase II activity in nitrogen mustard resistant human cells, Proc. Natl. Acad. Sci. USA 84 (1987) 7668–7671. [37] D.A. Boothman, D.K. Trask, A.B. Pardee, Inhibition of potentially lethal DNA damage repair in human tumor cells by bLapachone, an activator topoisomerase I1, Cancer Res. 49 (1989) 605–612. [38] S.R.R. Musk, G.G. Steel, Inhibitor of cellular recovery in human tumor cells by inhibitors of topoisomerases, Br. J. Cancer 62 (1990) 364–367. [39] T.K. Li, A.Y. Chen, C. Yu, Y. Mao, H. Wang, L.F. Liu, Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress, Genes Develop. 13 (1999) 1553–1560. [40] A. Lanza, S. Tornaletti, C. Rodolfo, M.C. Scanavini, A.M. Pedrini, Human DNA topoisomerase I-mediated cleavages stimulated by ultraviolet light-induced DNA damage, J. Biol. Chem. 271 (1996) 6978–6986. [41] P.R. Walker, C. Smith, T. Youdale, J. Lebane, J.F. Whitfield, M. Sikorska, Topoisomerase II reactive chemotherapeutic drugs induce apoptosis in thymocytes, Cancer Res. 51 (1991) 1078–1085. [42] S.H. Kaufmann, Cell death induced by topoisomerase targeted drugs: more questions than answers, Biochem. Biophys. Acta 1400 (1998) 195–211.
DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines Shailja Singh, B.S. Dwarakanath *, T. Lazar Mathew Department of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Brig SK Mazumdar Road, Timarpur, Delhi 110054, India Received 2 December 2003; received in revised form 25 June 2004; accepted 12 August 2004 Available online 8 October 2004
Abstract The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1–20 lM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 lM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2–3 folds at 5 lM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G2 blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity. 2004 Elsevier B.V. All rights reserved. Keywords: Clonogenic survival; Apoptosis; Micronuclei; Cell cycle perturbation; PLDR; Hoechst-33342; Aphidicolin
1. Introduction Development of primary therapeutic agents and adjuvants as well as strategies that selectively eliminate neoplastic cells has been the major focus of research in experimental oncology over the last few decades. In radiation therapy, the induction and repair of DNA lesions are central among the various cellular responses [1] that determine cellular lethality. Both these processes are influenced by a number of physico-chemical as well as biological parameters such as chromatin structure [2], tissue organization, presence of antioxidants, pool sizes *
Corresponding author. Fax: +91 11 2391 9509. E-mail address: [email protected] (B.S. Dwarakanath).
1011-1344/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2004.08.009
of repair enzymes and nucleotides, cell cycle status of the cells as well as optimal energy supply. Therefore, adjuvants that differentially modify the induction and/or repair of DNA damage in tumor and normal cells can significantly improve the therapeutic efficacy. The DNA ligands bisbenzimidazole derivative Hoechst-33258 and its ethoxy substituted analogue Hoechst-33342 form strong and non-covalent linkage with adenine and thymine rich regions in the minor groove of DNA [3,4]. The DNA bound ligands alter the degree of chromatin condensation besides inhibiting the activity of topoisomerase enzymes involved in the different DNA transactions, viz. DNA replication, transcription, gene expression and DNA repair [5,6]. The ligands have been shown to reduce radiation induced single and
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double strand breaks in aqueous DNA solutions as well as in cells [7–9]. Administration of these ligands prior to ionizing radiation exposure has also been found to marginally reduce radiation induced cytogenetic damage and cell death in vitro under certain conditions [9,10], and to protect whole body irradiated mice by reducing hematopoietic damage and enhancing animal survival [11]. However, in contrast to pre-irradiation treatment, the post irradiation treatment of cells with these ligands has been reported to enhance cell death [9]. While reduction in the induction of DNA damage has been fairly well established to be due to free radical scavenging and DNA radical quenching, the exact mechanisms underlying the radiosensitization action of Hoechst33342 are not yet fully understood. An understanding of the varying responses observed among different cells to low LET radiation is confounded by the effects of the ligand on the induction as well as repair of DNA damage, besides other damage dependent processes. Therefore, it was considered worthwhile to investigate the effects of the ligand on the cellular response to UV damage, which is predominantly caused by molecular excitation process rather than by free radical mediated damage as observed in the case of low linear energy transfer (LET) radiation. Using established human tumor cell lines, we have initiated systematic studies to investigate the effects of the ligand on the molecular (DNA) and cellular responses to UV induced damage. Results of the studies on the cellular responses to UV induced damage in two human glioma cell lines (U-87 and BMG-1) are presented in this communication. Treatment induced cellular responses such as cell cycle perturbation, growth inhibition, potential lethal damage recovery (PLDR), clonogenic cell death, cytogenetic damage and apoptosis were investigated.
2. Material and methods 2.1. Tumor cell lines
nyl]-2,5 0 -bi-1H-benzimidazole) trihydrochloride), HankÕs balanced salt solution (HBSS), DulbeccoÕs modified phosphate buffered saline (PBS), DulbeccoÕs modified eagleÕs medium (DMEM), fetal calf serum (FCS), N[2-hydroxyethyl] piperazine-N 0 -[2-ethanesulfonic acid] (HEPES) buffer, propidium iodide (PI), 4,6-diamidino2-phenyl indole (DAPI), Ribonuclease-A (RNase-A), trihydro-chloride and trypsin were obtained from Sigma Chemical Co., USA. All other chemicals used in the present study were of analytical grade from BDH, Glaxo laboratories (Qualigens), SRL, and E-Merck, India. 2.3. Ultraviolet light irradiation and treatment procedure A germicidal lamp (emitting most of its power at 254 nm) was used as a source of UV irradiation. Cells were irradiated at a distance of 30 cm from the UV lamp on ice. The incident dose rate measured under these conditions with a radiometer was 5.15 J m2 s1 (Model No. IL 1400 A, USA). Cells were incubated with varying concentrations (1– 20 lM) of Hoechst-33342 in HBSS at 37 C for 1 h, both before and after UV irradiation (26.75 J/m2). Following irradiation cells were incubated for different time interval at 37 C in complete media for studying different cellular responses. 2.4. Clonogenic survival assay After harvesting with 0.05% trypsin, 150–4000 (depending on the treatment) cells were plated 8–10 h before treating with varying concentrations (1–20 lM) of Hoechst-33342 in DMEM at 37 C for 1 h before and after UV irradiation. After the treatment, cells were incubated in dark under humidified, 5% CO2 atmosphere at 37 C for 8–10 days to allow colony formation. Colonies were fixed with methanol and stained with 1% crystal violet. Colonies of more than 50 cells were counted and the plating efficiency (PE) and the surviving fraction (SF) were calculated as described earlier [12].
Human cerebral glioma cell lines BMG-1 and U-87 used in the present studies were grown as monolayer cultures in DMEM with 5% fetal calf serum for BMG-1 cells and DMEM containing 10% FCS for U-87 cells and antibiotics i.e. penicillin (100 units/ml), streptomycin (50 lg/ ml) and nystatin (2 lg/ml). Stock cultures were passaged every third day after harvesting the cells with 0.05% trypsin and seeding 8 · 103 cells/cm2 in tissue culture flasks to maintain the cells in the exponential phase. All experiments were carried out in exponentially growing cells.
2.5. Potentially lethal damage recovery
2.2. Chemicals
After treatment, cells were incubated in growth medium for varying intervals of time, harvested by trypsinization and counted in a hemocytometer (adherent + floating). Cell proliferation was calculated by computing
The bisbenzimidazole derivative Hoechst-33342 (bis benzimide (2 0 -[4-ethoxyphenyl]-5-[4-methyl-1-piperazi-
Cultures were grown to a confluence and treated with Hoechst-33342 and UV without fresh medium supply. After treatment, cells were incubated in HBSS containing 1% serum for varying time intervals, harvested and plated with growth medium for the assessment of colony forming ability as described above. 2.6. Cell proliferation kinetics
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the increase in cell number and the cell proliferation index P, calculated as P = Nt/N0, where, Nt is the number of cells at time t, N0, the number of cells at the time of treatment. 2.7. Cell cycle perturbations Flow cytometric measurements of cellular DNA content were performed with the ethanol (70%) fixed cells using the intercalating DNA fluorochrome, PI as described earlier [13]. Briefly, the cells (0.5–1 · 106) were washed in PBS after the removal of ethanol and treatment with ribonuclease-A (200 lg/ml) for 30 min at 37 C. Subsequently, cells were stained with PI (50 lg/ml) in PBS. Measurements were made with a laser based (488 nm) flow cytometer (Facs Calibur; Beckton-Dickenson, USA) and data acquired using the Cell Quest software (Beckton-Dickenson, USA). Cell cycle analysis was performed using the Modfit program (BecktonDickenson, USA). 2.8. Micronuclei formation Air-dried slides containing acetic acid–methanol (1:3 V/V) fixed cells were stained with 2-aminophenylindoledihydrochloride (DAPI) (10 lg/ml in citric acid (0.01 M), disodium phosphate (0.45 M) buffer containing 0.05% Tween-20 detergent) as described earlier. Slides were examined under the fluorescence microscope using an UV excitation filter. Fluorescent nuclei were visualized using a blue emission filter. Cells containing micronuclei were counted from >1000 cells by employing the criteria of Countrymen and Heddle [14]. The fraction of cells containing micronuclei, called the M-fraction (%) was calculated as follows: M fraction ð%Þ ¼ N m =N t 100; where Nm is the number of cells with micronuclei and Nt is the total number of cells analyzed. Since, micronuclei formation is linked to cell proliferation, the micronuclei frequencies were normalized with respect to the cell numbers. 2.9. Detection of apoptotic cells Morphologically, marked condensation and margination of chromatin, fragmentation of nuclei and cell shrinkage characterize apoptotic cells and a good correlation between these morphological changes and DNA fragmentation (ladder) as hallmarks has been demonstrated [15]. The percentage of cells undergoing apoptosis was determined microscopically and by flow cytometry using PI and DAPI labeled cells. At least 1000 cells were counted and the percentage of apoptotic cells determined from slides prepared as described under Section 2.8 for micronuclei formation.
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In flow cytometric DNA analysis, the presence of a hypodiploid (sub G0/G1) population (with PI stained cells, as described for cell cycle analysis) is indicative of an apoptotic cell population. Measurements of cellular DNA content were made from PI stained, ethanol fixed cells as described in Section 2.7. Analysis of the hypodiploid population was performed using the Modfit program (Beckton-Dickenson, USA).
3. Results 3.1. Clonogenic cell survival A dose dependent decrease in SF was found by macro-colony assay (clonogenecity) in both BMG-1 and U87 cell lines and the extent of cell death was marginally higher in BMG-1 as compared to U-87 cells (Fig. 1). An UV dose of 26.75 J/m2 was found to be the isosurvival dose for the therapeutically relevant gamma ray dose of 2 Gy. Effects of Hoechst-33342 on UV (26.75 J/m2) induced cell death were studied as a function of the Hoechst-33342 concentration (1–20 lM). Pre and post-irradiation treatments with Hoechst-33342 enhanced the UV induced cell death in BMG-1 cells, in a concentration dependent manner (Fig. 1), suggesting an inhibition in the repair processes and or enhanced damage fixation. This was in contrast to our earlier observations with gamma irradiation, where a small decrease in cell death was noted when Hoechst-33342 was added 1 h before irradiation [9]. The extent of cell death was marginally higher when cells were treated with Hoechst-33342 immediately following irradiation as compared to pretreatment with Hoechst-33342 at all the concentrations. However, at higher concentrations of Hoechst-33342 (20 lM) the cytotoxic effects of the combined treatment (Hoechst-33342+UV) were synergistic and the increase in cell death was 5 fold (SF = 0.03) higher than the expected value from an additive effect (SF = 0.15) in BMG-1 cells. Similar results were obtained with U-87 cells (Fig. 1). In both the cell lines investigated, the extent of sensitization was marginally reduced (10–15%) when Hoechst-33342 (1 and 5 lM) was added at various time intervals following UV irradiation, implying that Hoechst-33342 interferes with the repair processing of UV-induced lesions (Fig. 2). To examine whether the Hoechst-33342 induced modification of UV toxicity in these asynchronously growing cultures was related to the status of DNA synthesis, we treated cells with the DNA polymerase inhibitor aphidicolin for 15 min prior to and during a subsequent exposure to Hoechst-33342. In the presence of 10 lM aphidicolin, the fraction of BrdU labeled cells (indicative of actively DNA synthesizing cells) decreased from approximately 45% to less than 2% in 5 min (data
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Fig. 1. Effects of Hoechst-33342 (added 1 h before or after) on the survival of UV irradiated (26.75 J/m2) exponentially growing human glioma cell lines BMG-1 and U-87 studied by macro colony assay. Inset UV dose response survival of BMG-1 and U-87 cells. Data presented are mean values (±SD) from at least five independent observations.
not shown). Under these conditions, the sensitivity of unirradiated cells to Hoechst-33342 was reduced by nearly 2 fold, whereas a 3–4 fold reduction was observed in UV irradiated cells (Fig. 3), implying that the Hoechst-33342 induced enhancement of UV cytotoxicity was highly dependent on the ongoing DNA synthesis. Treatment of cells with aphidicolin alone produced minimal cell killing (SF = 0.8) (Fig. 3).
Fig. 2. Effects of varying the time between UV irradiation (26.75 J/m2) and addition of Hoechst-33342 on the survival of human glioma cell lines BMG-1 and U-87. Data presented are mean values (±SD) from at least three independent observations.
crease in the SF over time, implying PLDR. Survival of cells treated with UV and Hoechst-33342 was significantly reduced in a concentration dependent manner (Fig. 3). After 4 h post-treatment incubation survival was reduced 10 folds as compared to immediate plating. Longer incubation did not result in additional cell killing (Fig. 4). 3.3. Cell proliferation
3.2. Potentially lethal damage recovery To study the effects of Hoechst-33342 on PLDR, confluent arrested cells were incubated in suboptimal growth conditions (1% serum + HBSS) for different time intervals following UV and combined treatments (1 and 5 lM Hoechst-33342+UV). Cells held under these conditions following UV irradiation showed a steady in-
The effects of Hoechst-33342 on the proliferation of exponentially growing cells were studied following UV irradiation by monitoring the kinetics of cell growth. A concentration dependent reduction in the rate of cell proliferation was observed (Fig. 5), with a lag period followed by a recovery from growth inhibition at a concentration of 5 lM. However, a cytostatic effect up to 48 h,
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Fig. 3. Effects of DNA synthesis inhibitor aphidicolin on Hoechst33342 induced modification of UV cytotoxicity in exponentially growing BMG-1 cells. Aphidicolin (10 lM) added 5 min before exposure of cells to Hoechst-33342 (20 lM) and UV irradiation (26.75 J/m2) was present for 1 h along with Hoechst-33342. Values are mean (±SD) of three observations from three independent experiments.
followed by a marginal increase in the cell number in both the cell lines was observed at 20 lM Hoechst33342 (Fig. 5). 3.4. Cell cycle perturbations To investigate the role of cell cycle perturbations after UV irradiation due to the bound ligand, cell cycle distributions were analyzed by cytofluorimetric measurements of cellular DNA contents at various time intervals following the treatment. UV irradiation transiently delayed cell cycle progression, with a significant accumulation in G2–M phase after 48 h post-treatment incubation (Fig. 6). This delay was further enhanced by Hoechst-33342 (5 lM) in both cell lines. While the G2–M block was transient in BMG-1 cells, it was nearly irreversible in U-87 cells even up to 72 h (Fig. 6). At a higher concentration of Hoechst-33342 (20 lM), a profound decrease in G2–M phase cells was observed in both cell lines with a concomitant increase in cell death possibly due to apoptosis as indicated by the appearance of cell population with a hypodiploid DNA content (Fig. 6). 3.5. Cytogenetic damage Mitotic death (linked to cytogenetic damage) and interphase death (apoptosis) together account for the cytotoxicity of many physicochemical agents including
Fig. 4. Effects of Hoechst-33342 on PLDR in BMG-1 and U-87 cell line studied by macro colony assay. Values are mean (±SD) of three observations from three independent experiments.
UV, although the relative contributions of the two death processes vary among the type of damaging agent. To investigate the modifications of UV-induced cytogenetic damage by Hoechst-33342, we studied the treatment-induced micronuclei formation in both cell lines. In UV irradiated cells, strand breaks generated during the excision repair process remaining unsealed lead to the formation of micronuclei in subsequent mitosis, and cells with micronuclei are found to be associated with loss of reproductive capacity [17]. Since cell proliferation influences treatment induced micronuclei expression, the data from kinetic studies up to 72 h post treatment were analyzed. The frequency of unirradiated cells with micronuclei was in the range of 1–2%, and treatment with 5 lM Hoechst-33342 did not induce a significant level of micronuclei formation in these cells (63%).
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sub G0/G1 population showed that UV and Hoechst33342 alone (5 and 20 lM) did not induce a significant level of apoptosis in both cell lines. However, the combined treatment significantly enhanced apoptosis in a time and concentration dependent manner in both the cell lines (Fig. 6). The frequency of apoptotic cells increased by nearly 50% following treatment with 20 lM Hoechst-33342 and UV at 72 h. The non-apoptotic population consisted of predominantly G1 and S and to a lesser extent of cells in G2–M phase cells (Fig. 6). Further, these results were supported by the morphological analysis of apoptosis (Fig. 8).
4. Discussion
Fig. 5. Effects of Hoechst-33342 (5 and 20 lM) on the proliferation of exponentially growing BMG-1 and U-87 cells. Cells were exposed to Hoechst-33342 for 1 h in HBSS immediately after UV irradiation. Data presented are mean values (±SD) from at least four independent observations. Where not shown, the SDÕs were less than the size of the symbols.
However, micronuclei frequency increased from 5% in UV irradiated cells to 12% with the combined treatment (5 lM Hoechst-33342+UV) at 48 h in BMG-1 cells following a decrease at 72 h. A comparable increase in U87 cells was also observed at 48 h, which however increased further at 72 h (Fig. 7). Due to extensive damage (interphase death), micronuclei expression could not be analyzed in cells treated with a higher concentration of Hoechst-33342 (20 lM) following UV irradiation. 3.6. Apoptosis The apoptotic mode of cell death in these cell lines was confirmed by microscopy and flow cytometry: the changes in cell morphology were characterized by typical chromatin condensation and nuclear fragmentation, with disruption of plasma membrane integrity and the appearance of a cell population with a hypodiploid DNA content. Flow-cytometric analysis of hypodiploid
Results of the present studies clearly demonstrate that both pre and post UV irradiation treatments with the DNA ligand Hoechst-33342 sensitizes cells in a concentration dependent manner, by increasing both the UV induced mitotic (linked to cytogenetic damage) and interphase (apoptosis) death. This is in contrast to ionizing radiation, where pre-irradiation Hoechst33342 treatment has been found to moderately decrease the lethality under certain condition [9]. Since UV irradiation causes mainly pyrimidine dimers due to direct UV absorption by DNA unlike the induction of DNA damage by low LET ionizing radiation via the generation of free radicals, the effects of Hoechst33342 on UV irradiated cells may be mainly due to its interference with repair and with the resolution of cleavable complexes. On the other hand, a net effect consequent to the alterations (mainly a reduction) in the damage induction as well as inhibition of repair processes determines the modification of cellular responses to ionizing radiation damage by Hoechst33342. In this respect, therefore studies on the effects of Hoechst-33342 on cellular responses to UV damage could be useful in elucidating its role in the biological response to radio-modification, besides the known effects of the ligand on radio-chemical aspects of damage induction [18]. In both the cell lines investigated here, the extent of sensitization was marginally reduced (10–15%) when Hoechst-33342 (5 lM) was added at various time intervals following UV irradiation (Fig. 2), implying that lesions whose processing is interfered by Hoechst-33342 are repaired to a certain extent with time. Hoechst33342 exhibits its cytotoxicity by interfering with the breakage-reunion reaction of DNA topoisomerases leading to the accumulation of intermediates termed Ôcleavable complexÕ [5,6]. At higher concentrations, the cytotoxic effects of the combination of Hoechst-33342 and UV were highly synergistic suggesting the possible interaction of lesions caused by both the agents i.e. UV and Hoechst-33342.
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Fig. 6. Flow cytometric DNA histograms from a typical experiment showing the effects of Hoechst-33342 on UV induced G2 block and apoptosis (arrow) in exponentially growing BMG-1 and U-87 cells.
Interestingly, under liquid holding conditions (HBSS + 1% serum), the DNA bound ligand not only inhibited the recovery from potentially lethal damage but also resulted in a time dependent increase in cell killing (Fig. 4). Fixation of many potentially lethal damage including part of the thymine dimers induced by UV irradiation under several conditions including those, which favour chromosome condensation [19] is expressed as enhanced chromosomal aberrations manifesting in mitotic death [20,21]. It appears therefore, that alterations in chromatin dynamics induced by the bound ligand [3,4] not only interferes in the repair, but also facilitates lesion fixation. Moreover, Hoechst-33342 remains bound to the cellular DNA for long intervals of time and stabilizes cleavable complex mediated DNA strand breaks, whose interaction with the primary UV lesion can also enhance lethality under these conditions. UV induced DNA lesions viz. thymine dimers and 6-4 photoproducts, block the DNA replication [22],
leading to the formation of DNA double strand breaks and chromosomal aberrations giving rise to acentric fragments because of nuclease attack at stalled replication forks [23,24]. When strand breaks generated during the repair of these lesions remain unligated, it results in chromosomal aberrations [14], which manifest as micronuclei in the subsequent mitosis. Micronuclei formation is therefore, one of the good indices to measure the residual DNA damage and loss of reproductive ability of cells [17]. The enhanced cell death due to Hoechst-33342 was also accompanied by a significant increase (2–3 folds at 5 lM Hoechst-33342) in UV induced micronuclei formation in both the cell lines (Fig. 7). This result suggests that the bound ligand profoundly inhibits the rejoining of strand breaks created during the excision repair (the major pathway for the repair of UV lesions), although it could interferes with the initial events of damage recognition and strand scission as
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Fig. 7. Effects of Hoechst-33342 (5 lM) on the kinetics of micronuclei expression following UV irradiation in exponentially growing BMG-1 and U-87 cells plotted as a function of time. Values are mean (±SD) of three independent observations.
well. Indeed inhibition of excision repair following UV induced DNA damage by the bound ligand has been observed (Shailja Singh et al., unpublished observations). However, the ligand may also induce a higher rate of faulty mitosis in damaged cells, which could partly contribute to the enhanced micronuclei observed. One of the characteristics of cellular response to UV irradiation is the accumulation of cells in S as well as G2–M phases of the cell cycle due to the inhibition of the replication by UV lesion, which was indeed observed in both the cell lines investigated (Fig. 6). In case of Hoechst-33342 bound cells, the transient accumulation of cells in G2–M phases of cell cycle is likely to be due to the presence of low levels of cleavable complexes (protein linked DNA breaks) as well as interference in chromosome segregation on account of topoisomerase II inhibition [25]. Cell cycle delay arises as an account of the operational functional checkpoints at G1–S and G2–M transition and the ex-
Fig. 8. Effect of Hoechst-33342 (20 lM) on UV induced apoptosis studied as a function of post-irradiation time in BMG-1 and U-87 cells. Data presented are mean values (±SD) from at least three independent observations.
tent of delay is related to the level of DNA damage [26]. The enhanced delay observed in Hoechst-33342 bound cells, therefore, indicates the presence of a higher level of DNA damage following UV irradiation. Since the cytotoxic effects of Hoechst-33342 has been found to be significantly higher in the S phase cells than in the G1 and G2–M phases of cell cycle [27], prolonged S phase by UV irradiation seems to potentiate the cytotoxic effects of the combined treatment (Hoechst-33342+UV) by inducing a larger cell population to be in the sensitive state for Hoechst33342 induced cell killing. The marked S phase specificity is thought to result from the interaction of topo I cleavable complex with replication fork resulting in the generation of lethal DNA double strand breaks like in case of the well known topoisomerase I inhibitor camptothecin [16,28]. Consistent with this hypothesis, we have observed a reduction in the Hoechst-33342 induced UV sensitization with DNA a polymerase inhibitor aphidicolin in BMG-1 cells (Fig. 3). In fact, the induction of double strand break in replicating DNA
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due the collision of replication fork with topoisomerase I involved cleavable complex has been proposed as a cause for the S phase specific lethal effects of topoisomerase I inhibitors [29]. Loss of clonogenicity following the induction of DNA damage could arise either due to the mitotic death and/or due to apoptosis depending on the cell type. Since the principle mechanisms of apoptosis differ in several ways from that of micronuclei formation, these are independent predictors of cellular sensitivity [30]. Nearly 50% increase in the UV induced apoptosis observed in both the cell lines at higher concentrations of Hoechst-33342 (20 lM) imply that the enhancement in cell death is significantly mediated through enhanced apoptosis as well (Fig. 6). Further, it was also found that non-apoptotic population consisted of predominantly G1 and S phase cells to a lesser extent G2–M phase cells. Programmed cell death (apoptosis) induced by UV-C light in various cell types has been proposed to be a consequence of receptor activation [31], as well as by p53 activation due to the UV induced DNA lesions stalling the transcription machinery [32,33]. Recently it has been also proposed that double strand breaks are the ultimate trigger of UV-C induced apoptosis [34]. Lesions induced by UV can be reversed by a number of repair pathways, which involve several gene products viz. endonucleases or exonucleases, polymerases and ligases [35,36]. Important roles for topoisomerase can be envisioned in these repair steps, and circumstantial evidences currently available indicate the participation of topoisomerase I and II in the repair of various forms of the DNA damage [36–38]. Contrary to its role in the repair of DNA damage, topoisomerases have been implicated in the generation of potentially lethal DNA breaks by interaction with UV induced DNA damage as well as with topoisomerase specific poisons [39,40]. Further, topoisomerase cleavable complex mediated breaks have been shown to induce apoptosis [41,42]. Therefore, it appears that inhibition in the repair of UV induced lesions as well as formation cleavable complex mediated, S phase dependent and/or independent strand breaks are to a very great extent responsible for the enhanced apoptosis in cells following the combined treatment with Hoechst33342 and UV-C.
Acknowledgements We thank Maj. Gen. T. Ravindranath Director, INMAS for his constant support and encouragement. Useful discussion during the initial part of this work with Prof. Viney Jain, Wright State University, USA is gratefully acknowledged. We thank J.S. Adhikari, Dr Seema
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Gupta and Rohit Mathur for helpful discussions and technical support.
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