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Caffeine modulates heat shock induced apoptosis in the human promyelocytic leukemia cell line HL-60
Cancer Letters 121 (1997) 1–6
Caffeine modulates heat shock induced apoptosis in the human promyelocytic leukemia cell line HL-60 Bryan S. Poe, Kim L. O’Neill* Department of Microbiology, Brigham Young University, Provo, UT 84602, USA Received 29 April 1997; received in revised form 22 May 1997; accepted 22 May 1997
Abstract The ability of caffeine to modulate hyperthermia induced apoptosis was investigated in the human promyelocytic cell line HL60. Mild hyperthermia has been shown to be a strong inducer of apoptosis in many cell lines of lymphoblastoid lineage. In this investigation HL60 cells were simultaneously exposed to caffeine (concentrations ranging from 0 to 20 mM), and a brief hyperthermic treatment (43°C) for 1 h and then allowed a recovery time of 12 h. Approximately 50% of a cell population receiving the hyperthermia treatment died by apoptosis within 12 h, as determined by the comet assay, whereas cells that received concomitant treatments of caffeine with heat shock displayed an apparent suppression of apoptotic induction and enhanced cell survival. 1997 Elsevier Science Ireland Ltd. Keywords: Heat shock; Apoptosis; Caffeine; Comet assay; Single cell gel electrophoresis
1. Introduction The term apoptosis, first published by Kerr et al. [1] in their seminal paper, is used to denote a programmed mode of cellular ‘suicide’. During the course of this program there are several key events that occur, such as loss of cell volume, membrane boiling or blebbing and, most notably, a number of striking changes in the nucleus [2,3]. At the nuclear level, chromatin becomes densely packed around the periphery and genomic DNA is degraded in at least two stages. First, the DNA is cleaved into 50–300 kb fragments which are then subsequently degraded into smaller pieces with lengths of oligonucleosomal multiples. These pieces, when extracted from the cell and subjected to agarose * Corresponding author.
gel electrophoresis, display a ladder-like appearance indicative of nucleosomal size fragments associated with apoptotic DNA damage [4]. Extensive DNA fragmentation is one of the most common endpoints used in assaying for apoptosis; as a consequence, a number of techniques have been devised to detect apoptotic DNA damage. Of the many techniques described in the literature the comet assay, originally introduced by Ostling and Johansen [5] is perhaps one of the most sensitive tools available for detecting DNA damage in individual cells [6]. Briefly, cells embedded in agarose are lysed and their DNA is subsequently subjected to electrophoresis. The differential migration of DNA fragments away from the nuclear ‘head’ form a fluorescent ‘tail’, producing DNA images resembling comets. Because severe DNA damage is a common
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0286-3
2
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
endpoint in many detection systems, the comet assay has proven to be an extremely sensitive and reliable method of choice in several studies [7–9]. Caffeine (3,7-dihydro-1,3,7,-trimethyl-1H-purine6,6-dione) is a compound which exerts multiple effects on target cells both in vivo and in vitro. Its pharmacological effects are highly dependent on both drug concentration and the nature of the target cells. Many studies have explored the pharmacological activity of caffeine to potentiate cytotoxicity in cells concomitantly exposed to DNA damaging agents such as alkylating agents, ionizing radiation and cisplatinum analogues [10]. This potentiating phenomenon is thought to be a result of the suppressive activities of caffeine towards DNA repair mechanisms [11]. The logical consequences of DNA repair suppression are eventual cell death due to lethal genetic lesions, genic mutations in daughter cells due to unfaithful replication of damaged parental chromosomes, or mutations arising from inaccurate repair performed by a compromised genetic repair apparatus. In contrast to caffeine’s role as a potentiator, it has been demonstrated that in certain instances it also possesses a protective activity which can diminish or even abrogate the cytocidal or cytostatic effects of several drugs known to interact with DNA, such as camptothecin, VM-26, TPT and doxorubicin [10]. Depending on concentration and cell type, these compounds exert strong cell cycle effects or can induce apoptosis as a common mode of cell death [11]. The drugs that have been shown to have their activities negatively modulated by caffeine have three major themes in common: (i) they tend to be flat aromatic molecules; (ii) they intercalate DNA; and (iii) they are DNA topoiomerase inhibitors [12]. Based on the biophysical interactions of these types of drugs and caffeine in solution, it has been proposed that the protective mechanism of caffeine involves the formation of stackable complexes with the drugs, reducing the amount of free drug available for cellular interactions. The formation of these complexes has been suggested to involve the aromatic nature of the drugs rather than with any perturbation of the DNA–drug interactions or enzyme involvement [10]. The intent of this study has been to explore the protective relationship of caffeine with an apoptogenic agent that would not overtly affect the biologi-
cal activity of caffeine in vitro. For this reason a well established heat shock model was chosen for apoptotic induction. Our results from this study indicate that an apoptotic response could be significantly diminished using caffeine as a sole modulating agent.
2. Materials and methods 2.1. Chemicals Caffeine was purchased from Sigma (St. Louis, MO). The stock solution of caffeine was prepared at a concentration of 200 mM in distilled water. Working solutions were prepared immediately before use by diluting stock solution into cell culture media. 2.2. Cell culture HL-60 myologenous leukemia cells were maintained in RPMI 1640 culture media (HyClone, Logan, UT) supplemented with 10% bovine calf serum (HyClone), 2 mM l-glutamine, 100 mg/ml streptomycin and 100 units/ml penicillin (Sigma, St. Louis, MO). The cells were split every third day and diluted 1:2 1 day before each experiment was performed. Cell densities in culture did not exceed 5 × 105 cells/ml. 2.3. Hyperthermia treatment Just prior to the hyperthermia treatment, 1–2 × 105 cells were removed from culture, washed in phosphate buffered saline (PBS) and resuspended in fresh whole media in 1.5 ml micro centrifuge tubes. The tubes containing the target cells were then placed in a waterbath for 1 h at various temperatures (37, 40, 43 and 45°C). The amount of recovery time after exposure was varied from 0 to 24 h after the time of initial exposure. 2.4. Single cell gel electrophoresis The single cell gel assay was performed essentially as described previously with some minor modifications [13]. Following hyperthermic treatment, low melting point agarose (Sigma) was added to tubes containing 2.5 × 104 –3.0 × 104 cells suspended in
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
3
and Dr. P.L. Olive. The software analyzes the fluorescent image captured by the camera and transforms the pixel intensity found in the head and tail of the ‘comets’ into a measure referred to as ‘tail moment’ [14]. Tail moment is described as the product of the tail length and the intensity of the DNA in the tail of the comet.
3. Results and discussion
Fig. 1. Thirty-six hour time course of heat shock induced apoptosis in HL-60 cells. Cells were incubated at 43°C for 1 h and allowed a course of recovery times ranging from 0 to 36 h. Apoptoses were first detected at 6 h post-heat shock and plateaued at 12 h.
phosphate buffered saline (PBS) with a final agarose concentration of 0.75%. The contents of the tubes were then carefully layered onto fully frosted microscope slides and allowed to gel on an ice cold surface. The slides were then placed in a bath of alkaline lysis buffer, consisting of 1 M NaCl and 0.5% N-lauroyl sarcosine (pH adjusted to 12.3 with NaOH), for 1 h at room temperature. Slides were then washed free of salt by immersion in an alkaline running buffer (2 mM EDTA and 0.03 M NaOH) for 1 h. The wash step is essential since salt retards migration in the following electrophoresis step. Next the slides were placed in a horizontal electrophoresis chamber and electrophoresed in a fresh solution of alkaline running buffer for 15 min at 1.0 V/cm. Following electrophoresis the slides were neutralized in distilled water for 10 min and stained for 30 min in a staining bath of 2.5 mg/ml propidium iodide. The comets were analyzed using a Carl Zeiss epifluorescent microscope and integrated CCD camera/computer image analysis system. Two hundred nuclei were randomly scored from each slide and each experiment was performed at least three times. In order to use this assay to discriminate apoptotic DNA damage from necrotic damage additional analyses were performed using an analytical software package, kindly provided by Dr. R.E. Durant
The cellular response to hyperthermia has been established as a reliable method for inducing apoptotic cell death, with apoptoses resulting from lethal heat shock observable shortly after treatment [14] (Fig. 1). We have previously shown that there is a distinct apoptotic temperature window in which the thermal hit will preferentially induce apoptosis over necrosis in certain cell populations [15]. In order to establish a reliable intensity and duration of the thermal hit, which would induce apoptosis preferentially over necrosis, we used several different temperatures and recovery periods. By changing both the recovery times and the temperatures we established a reliable level of apoptotic induction (Figs. 1 and 2). By using the comet assay we were able to readily identify apop-
Fig. 2. The shift from apoptosis to necrosis as a function of increased heat load. Cells were incubated under hyperthermic conditions ranging from 37°C to 50°C. Apoptoses were subsequently detected and viabilities determined by Trypan Blue exclusion.
4
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
totic nuclei based on characteristic morphological changes attributable to extensive DNA damage (Fig. 3a–c). We have shown that an increase in heatload on cells that are normally sensitive to heat shock induced apoptosis is sufficient to change the mode of death
Fig. 4. Assessment of the cytotoxic effects of caffeine on HL-60 cells. Cells were exposed to the indicated concentrations of caffeine in a 96-well culture plate for 24 h at which point viabilities were determined using the Trypan Blue exclusion method.
from apoptosis to necrosis [9]. By varying the temperature from 37 to 50°C a decrease in viability is seen at approximately 44°C with the apoptotic fraction peaking at 43°C (Fig. 2). After assessing a time course over 36 h we found that the cells exposed at the optimal apoptotic temperature (43°C for 1 h) dis-
Fig. 3. Morphological results of the comet assay. (a) The nondegraded DNA in the nucleus of the control cell remains intact when subjected to electrophoresis. (b) The highly fragmented DNA in the nucleus of the apoptotic cell migrates away from the nuclear head as a function of the severity of cleavage. (c) The moderately degraded DNA in the nucleus of a necrotic cell is morphologically distinct from apoptotic fragmentation.
Fig. 5. Caffeine inhibits heat shock induced apoptosis in HL-60 cells.
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
played a gradual increase in cell death that peaked at 12 h post-exposure and slowly declined from this plateau through a 36 h time point (Fig. 1). From these data we choose a thermal hit of 43°C and a recovery period of 12 h as this gave us a level of induction that was easily distinguishable from background apoptoses and resulted in little or no necrotic cell death attributable to an excessive heat load. We initially assessed the potential cytotoxic effects of caffeine on HL60 cells to qualify a permissible working concentration range. Using Trypan Blue exclusion, we determined cell viability at a small concentration range from 0 to 20 mM (Fig. 4). We observed no significant reduction in viability nor an increase in the apoptotic index above the background control levels of unexposed cells at concentrations between 0 and 10 mM after 24 h. Above these concentrations, a dose dependent reduction in cell viability was seen without an increase in apoptosis. Accordingly, we choose only those concentrations of caffeine for which neither loss of viability nor cytopathological effects were seen. Next we exposed target cells to a concomitant treatment of hyperthermia and caffeine. The data accumulated indicate that caffeine has a protective effect in apoptotic induction (Fig. 5). A small range of caffeine
Fig. 6. Caffeine induces cell death and subsequent DNA strand breakage characteristic of passive necrotic death but does not induce apoptotic DNA fragmentation based on tail moment analysis.
5
Fig. 7. Cells treated with caffeine show a higher survival number after hyperthermic treatment. Cells were given a concomitant treatment of caffeine (various concentrations) and heat shock. Viabilities were assessed 24 h after treatment using the Trypan Blue exclusion method.
concentrations was assayed using the alkaline comet assay. A protective window in the concentration range was observed to occur between 2.5 and 10 mM with no unusual effects seen on either side of this concentration range. At caffeine concentrations greater that 20 mM we saw much secondary DNA fragmentation which was not associated with apoptotic events but more in line with a necrotic process as determined by tail moment analysis of this cell fraction. We have previously shown that tail moment is sufficient to discriminate apoptotic from necrotic cell fractions [9] (Fig. 6). Additionally, cell viabilities were assessed 24 h after the heat shock and caffeine regimen (at which time apoptotic cells lose the ability to exclude Trypan Blue) (Fig. 7). These results together indicate a higher viability for the caffeine heat shock treated cells over cells receiving heat shock alone. The work presented here is preliminary in nature but does allow some insights into the mechanisms of this protective phenomenon. Owing to the nature of the inducer used in this model, our data suggest that the possible sequestration of apoptogenic compounds via the formation of ‘stackable’ complexes with caffeine may not be the only mechanism involved in caffeine related inhibition of apoptosis.
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B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
References [1] J.F. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics, Br. J. Cancer 26 (1972) 239–257. [2] H. Steller, Mechanisms and genes of cellular suicide, Science 267 (1995) 1445–1449. [3] E. White, Life, death and the pursuit of apoptosis, Genes Dev. 10 (1996) 1–15. [4] A.H. Wyllie, Death from the inside out: an overview, Phil. Trans. R. Soc. London 345 (1995) 237–241. ¨ stling, J.P. Johanson, Microelectrophoretic study of [5] O. O radiation induced DNA damage in individual mammalian cells, Biochem. Biophys. Res. Commun. 123 (1984) 291– 298. [6] D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutat. Res. 339 (1995) 37–59. [7] P.L. Olive, G. Frazer, J.P. Banath, Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay, Radiat. Res. 136 (1993) 130–136. [8] P.L. Olive, J.P. Banath, Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent, Exp. Cell Res. 221 (1995) 19–26. [9] D.W. Fairbairn, D.K. Walburger, J.J. Fairbairn, K.L. O’Neill, Key morphological changes and DNA strand breaks in human
[10]
[11]
[12]
[13]
[14]
[15]
lymphoid cells: discriminating apoptosis from necrosis, Scanning 18 (1996) 407–416. F. Traganos, J. Kapuscinski, J. Gong, B. Ardelt, R. Darzynkiewicz, Z. Darzynkiewicz, Caffeine prevents apoptosis and cell cycle effects induced by camptothecin or topotecan in HL-60 cells, Cancer Res. 53 (1993) 4613–4618. N. Shinomiya, M. Shinomiya, H. Wakiyama, Y. Katsura, M. Rokutanda, Enhancement of CDDP cytotoxicity by caffeine is characterised by apoptotic cell death, Exp. Cell Res. 210 (1994) 236–242. W. Gorczyca, J. Gong, B. Ardelt, F. Traganos, Z. Darzynkiewicz, The cell cycle related differences in susceptibility of HL-60 cell to apoptosis induced by various antitumor agents, Cancer Res. 53 (1993) 3186–3192. P.L. Olive, J.P. Bana´th, C.D. Fjell, DNA strand breakage and DNA structure influence staining with propidium iodide using the comet assay, Cytometry 16 (1994) 305–312. J.J. Fairbairn, M.W. Khan, K.J. Ward, B.W. Loveridge, D.W. Fairbairn, K.L. O’Neill, Induction of apoptotic cell DNA fragmentation in human cells after treatment with hyperthermia, Cancer Lett. 89 (1995) 183–188. D.W. Fairbairn, K.L. O’Neill, Necrotic DNA degradation mimics apoptotic nucleosomal fragmentation comet tail length, In Vitro Cell Dev. Biol. 31 (1994) 171–173.
Caffeine modulates heat shock induced apoptosis in the human promyelocytic leukemia cell line HL-60 Bryan S. Poe, Kim L. O’Neill* Department of Microbiology, Brigham Young University, Provo, UT 84602, USA Received 29 April 1997; received in revised form 22 May 1997; accepted 22 May 1997
Abstract The ability of caffeine to modulate hyperthermia induced apoptosis was investigated in the human promyelocytic cell line HL60. Mild hyperthermia has been shown to be a strong inducer of apoptosis in many cell lines of lymphoblastoid lineage. In this investigation HL60 cells were simultaneously exposed to caffeine (concentrations ranging from 0 to 20 mM), and a brief hyperthermic treatment (43°C) for 1 h and then allowed a recovery time of 12 h. Approximately 50% of a cell population receiving the hyperthermia treatment died by apoptosis within 12 h, as determined by the comet assay, whereas cells that received concomitant treatments of caffeine with heat shock displayed an apparent suppression of apoptotic induction and enhanced cell survival. 1997 Elsevier Science Ireland Ltd. Keywords: Heat shock; Apoptosis; Caffeine; Comet assay; Single cell gel electrophoresis
1. Introduction The term apoptosis, first published by Kerr et al. [1] in their seminal paper, is used to denote a programmed mode of cellular ‘suicide’. During the course of this program there are several key events that occur, such as loss of cell volume, membrane boiling or blebbing and, most notably, a number of striking changes in the nucleus [2,3]. At the nuclear level, chromatin becomes densely packed around the periphery and genomic DNA is degraded in at least two stages. First, the DNA is cleaved into 50–300 kb fragments which are then subsequently degraded into smaller pieces with lengths of oligonucleosomal multiples. These pieces, when extracted from the cell and subjected to agarose * Corresponding author.
gel electrophoresis, display a ladder-like appearance indicative of nucleosomal size fragments associated with apoptotic DNA damage [4]. Extensive DNA fragmentation is one of the most common endpoints used in assaying for apoptosis; as a consequence, a number of techniques have been devised to detect apoptotic DNA damage. Of the many techniques described in the literature the comet assay, originally introduced by Ostling and Johansen [5] is perhaps one of the most sensitive tools available for detecting DNA damage in individual cells [6]. Briefly, cells embedded in agarose are lysed and their DNA is subsequently subjected to electrophoresis. The differential migration of DNA fragments away from the nuclear ‘head’ form a fluorescent ‘tail’, producing DNA images resembling comets. Because severe DNA damage is a common
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0286-3
2
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
endpoint in many detection systems, the comet assay has proven to be an extremely sensitive and reliable method of choice in several studies [7–9]. Caffeine (3,7-dihydro-1,3,7,-trimethyl-1H-purine6,6-dione) is a compound which exerts multiple effects on target cells both in vivo and in vitro. Its pharmacological effects are highly dependent on both drug concentration and the nature of the target cells. Many studies have explored the pharmacological activity of caffeine to potentiate cytotoxicity in cells concomitantly exposed to DNA damaging agents such as alkylating agents, ionizing radiation and cisplatinum analogues [10]. This potentiating phenomenon is thought to be a result of the suppressive activities of caffeine towards DNA repair mechanisms [11]. The logical consequences of DNA repair suppression are eventual cell death due to lethal genetic lesions, genic mutations in daughter cells due to unfaithful replication of damaged parental chromosomes, or mutations arising from inaccurate repair performed by a compromised genetic repair apparatus. In contrast to caffeine’s role as a potentiator, it has been demonstrated that in certain instances it also possesses a protective activity which can diminish or even abrogate the cytocidal or cytostatic effects of several drugs known to interact with DNA, such as camptothecin, VM-26, TPT and doxorubicin [10]. Depending on concentration and cell type, these compounds exert strong cell cycle effects or can induce apoptosis as a common mode of cell death [11]. The drugs that have been shown to have their activities negatively modulated by caffeine have three major themes in common: (i) they tend to be flat aromatic molecules; (ii) they intercalate DNA; and (iii) they are DNA topoiomerase inhibitors [12]. Based on the biophysical interactions of these types of drugs and caffeine in solution, it has been proposed that the protective mechanism of caffeine involves the formation of stackable complexes with the drugs, reducing the amount of free drug available for cellular interactions. The formation of these complexes has been suggested to involve the aromatic nature of the drugs rather than with any perturbation of the DNA–drug interactions or enzyme involvement [10]. The intent of this study has been to explore the protective relationship of caffeine with an apoptogenic agent that would not overtly affect the biologi-
cal activity of caffeine in vitro. For this reason a well established heat shock model was chosen for apoptotic induction. Our results from this study indicate that an apoptotic response could be significantly diminished using caffeine as a sole modulating agent.
2. Materials and methods 2.1. Chemicals Caffeine was purchased from Sigma (St. Louis, MO). The stock solution of caffeine was prepared at a concentration of 200 mM in distilled water. Working solutions were prepared immediately before use by diluting stock solution into cell culture media. 2.2. Cell culture HL-60 myologenous leukemia cells were maintained in RPMI 1640 culture media (HyClone, Logan, UT) supplemented with 10% bovine calf serum (HyClone), 2 mM l-glutamine, 100 mg/ml streptomycin and 100 units/ml penicillin (Sigma, St. Louis, MO). The cells were split every third day and diluted 1:2 1 day before each experiment was performed. Cell densities in culture did not exceed 5 × 105 cells/ml. 2.3. Hyperthermia treatment Just prior to the hyperthermia treatment, 1–2 × 105 cells were removed from culture, washed in phosphate buffered saline (PBS) and resuspended in fresh whole media in 1.5 ml micro centrifuge tubes. The tubes containing the target cells were then placed in a waterbath for 1 h at various temperatures (37, 40, 43 and 45°C). The amount of recovery time after exposure was varied from 0 to 24 h after the time of initial exposure. 2.4. Single cell gel electrophoresis The single cell gel assay was performed essentially as described previously with some minor modifications [13]. Following hyperthermic treatment, low melting point agarose (Sigma) was added to tubes containing 2.5 × 104 –3.0 × 104 cells suspended in
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
3
and Dr. P.L. Olive. The software analyzes the fluorescent image captured by the camera and transforms the pixel intensity found in the head and tail of the ‘comets’ into a measure referred to as ‘tail moment’ [14]. Tail moment is described as the product of the tail length and the intensity of the DNA in the tail of the comet.
3. Results and discussion
Fig. 1. Thirty-six hour time course of heat shock induced apoptosis in HL-60 cells. Cells were incubated at 43°C for 1 h and allowed a course of recovery times ranging from 0 to 36 h. Apoptoses were first detected at 6 h post-heat shock and plateaued at 12 h.
phosphate buffered saline (PBS) with a final agarose concentration of 0.75%. The contents of the tubes were then carefully layered onto fully frosted microscope slides and allowed to gel on an ice cold surface. The slides were then placed in a bath of alkaline lysis buffer, consisting of 1 M NaCl and 0.5% N-lauroyl sarcosine (pH adjusted to 12.3 with NaOH), for 1 h at room temperature. Slides were then washed free of salt by immersion in an alkaline running buffer (2 mM EDTA and 0.03 M NaOH) for 1 h. The wash step is essential since salt retards migration in the following electrophoresis step. Next the slides were placed in a horizontal electrophoresis chamber and electrophoresed in a fresh solution of alkaline running buffer for 15 min at 1.0 V/cm. Following electrophoresis the slides were neutralized in distilled water for 10 min and stained for 30 min in a staining bath of 2.5 mg/ml propidium iodide. The comets were analyzed using a Carl Zeiss epifluorescent microscope and integrated CCD camera/computer image analysis system. Two hundred nuclei were randomly scored from each slide and each experiment was performed at least three times. In order to use this assay to discriminate apoptotic DNA damage from necrotic damage additional analyses were performed using an analytical software package, kindly provided by Dr. R.E. Durant
The cellular response to hyperthermia has been established as a reliable method for inducing apoptotic cell death, with apoptoses resulting from lethal heat shock observable shortly after treatment [14] (Fig. 1). We have previously shown that there is a distinct apoptotic temperature window in which the thermal hit will preferentially induce apoptosis over necrosis in certain cell populations [15]. In order to establish a reliable intensity and duration of the thermal hit, which would induce apoptosis preferentially over necrosis, we used several different temperatures and recovery periods. By changing both the recovery times and the temperatures we established a reliable level of apoptotic induction (Figs. 1 and 2). By using the comet assay we were able to readily identify apop-
Fig. 2. The shift from apoptosis to necrosis as a function of increased heat load. Cells were incubated under hyperthermic conditions ranging from 37°C to 50°C. Apoptoses were subsequently detected and viabilities determined by Trypan Blue exclusion.
4
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
totic nuclei based on characteristic morphological changes attributable to extensive DNA damage (Fig. 3a–c). We have shown that an increase in heatload on cells that are normally sensitive to heat shock induced apoptosis is sufficient to change the mode of death
Fig. 4. Assessment of the cytotoxic effects of caffeine on HL-60 cells. Cells were exposed to the indicated concentrations of caffeine in a 96-well culture plate for 24 h at which point viabilities were determined using the Trypan Blue exclusion method.
from apoptosis to necrosis [9]. By varying the temperature from 37 to 50°C a decrease in viability is seen at approximately 44°C with the apoptotic fraction peaking at 43°C (Fig. 2). After assessing a time course over 36 h we found that the cells exposed at the optimal apoptotic temperature (43°C for 1 h) dis-
Fig. 3. Morphological results of the comet assay. (a) The nondegraded DNA in the nucleus of the control cell remains intact when subjected to electrophoresis. (b) The highly fragmented DNA in the nucleus of the apoptotic cell migrates away from the nuclear head as a function of the severity of cleavage. (c) The moderately degraded DNA in the nucleus of a necrotic cell is morphologically distinct from apoptotic fragmentation.
Fig. 5. Caffeine inhibits heat shock induced apoptosis in HL-60 cells.
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
played a gradual increase in cell death that peaked at 12 h post-exposure and slowly declined from this plateau through a 36 h time point (Fig. 1). From these data we choose a thermal hit of 43°C and a recovery period of 12 h as this gave us a level of induction that was easily distinguishable from background apoptoses and resulted in little or no necrotic cell death attributable to an excessive heat load. We initially assessed the potential cytotoxic effects of caffeine on HL60 cells to qualify a permissible working concentration range. Using Trypan Blue exclusion, we determined cell viability at a small concentration range from 0 to 20 mM (Fig. 4). We observed no significant reduction in viability nor an increase in the apoptotic index above the background control levels of unexposed cells at concentrations between 0 and 10 mM after 24 h. Above these concentrations, a dose dependent reduction in cell viability was seen without an increase in apoptosis. Accordingly, we choose only those concentrations of caffeine for which neither loss of viability nor cytopathological effects were seen. Next we exposed target cells to a concomitant treatment of hyperthermia and caffeine. The data accumulated indicate that caffeine has a protective effect in apoptotic induction (Fig. 5). A small range of caffeine
Fig. 6. Caffeine induces cell death and subsequent DNA strand breakage characteristic of passive necrotic death but does not induce apoptotic DNA fragmentation based on tail moment analysis.
5
Fig. 7. Cells treated with caffeine show a higher survival number after hyperthermic treatment. Cells were given a concomitant treatment of caffeine (various concentrations) and heat shock. Viabilities were assessed 24 h after treatment using the Trypan Blue exclusion method.
concentrations was assayed using the alkaline comet assay. A protective window in the concentration range was observed to occur between 2.5 and 10 mM with no unusual effects seen on either side of this concentration range. At caffeine concentrations greater that 20 mM we saw much secondary DNA fragmentation which was not associated with apoptotic events but more in line with a necrotic process as determined by tail moment analysis of this cell fraction. We have previously shown that tail moment is sufficient to discriminate apoptotic from necrotic cell fractions [9] (Fig. 6). Additionally, cell viabilities were assessed 24 h after the heat shock and caffeine regimen (at which time apoptotic cells lose the ability to exclude Trypan Blue) (Fig. 7). These results together indicate a higher viability for the caffeine heat shock treated cells over cells receiving heat shock alone. The work presented here is preliminary in nature but does allow some insights into the mechanisms of this protective phenomenon. Owing to the nature of the inducer used in this model, our data suggest that the possible sequestration of apoptogenic compounds via the formation of ‘stackable’ complexes with caffeine may not be the only mechanism involved in caffeine related inhibition of apoptosis.
6
B.S. Poe, K.L. O’Neill / Cancer Letters 121 (1997) 1–6
References [1] J.F. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics, Br. J. Cancer 26 (1972) 239–257. [2] H. Steller, Mechanisms and genes of cellular suicide, Science 267 (1995) 1445–1449. [3] E. White, Life, death and the pursuit of apoptosis, Genes Dev. 10 (1996) 1–15. [4] A.H. Wyllie, Death from the inside out: an overview, Phil. Trans. R. Soc. London 345 (1995) 237–241. ¨ stling, J.P. Johanson, Microelectrophoretic study of [5] O. O radiation induced DNA damage in individual mammalian cells, Biochem. Biophys. Res. Commun. 123 (1984) 291– 298. [6] D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutat. Res. 339 (1995) 37–59. [7] P.L. Olive, G. Frazer, J.P. Banath, Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay, Radiat. Res. 136 (1993) 130–136. [8] P.L. Olive, J.P. Banath, Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent, Exp. Cell Res. 221 (1995) 19–26. [9] D.W. Fairbairn, D.K. Walburger, J.J. Fairbairn, K.L. O’Neill, Key morphological changes and DNA strand breaks in human
[10]
[11]
[12]
[13]
[14]
[15]
lymphoid cells: discriminating apoptosis from necrosis, Scanning 18 (1996) 407–416. F. Traganos, J. Kapuscinski, J. Gong, B. Ardelt, R. Darzynkiewicz, Z. Darzynkiewicz, Caffeine prevents apoptosis and cell cycle effects induced by camptothecin or topotecan in HL-60 cells, Cancer Res. 53 (1993) 4613–4618. N. Shinomiya, M. Shinomiya, H. Wakiyama, Y. Katsura, M. Rokutanda, Enhancement of CDDP cytotoxicity by caffeine is characterised by apoptotic cell death, Exp. Cell Res. 210 (1994) 236–242. W. Gorczyca, J. Gong, B. Ardelt, F. Traganos, Z. Darzynkiewicz, The cell cycle related differences in susceptibility of HL-60 cell to apoptosis induced by various antitumor agents, Cancer Res. 53 (1993) 3186–3192. P.L. Olive, J.P. Bana´th, C.D. Fjell, DNA strand breakage and DNA structure influence staining with propidium iodide using the comet assay, Cytometry 16 (1994) 305–312. J.J. Fairbairn, M.W. Khan, K.J. Ward, B.W. Loveridge, D.W. Fairbairn, K.L. O’Neill, Induction of apoptotic cell DNA fragmentation in human cells after treatment with hyperthermia, Cancer Lett. 89 (1995) 183–188. D.W. Fairbairn, K.L. O’Neill, Necrotic DNA degradation mimics apoptotic nucleosomal fragmentation comet tail length, In Vitro Cell Dev. Biol. 31 (1994) 171–173.