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Apoptosis and p53 protein expression in human hepatoma cells induced by etoposide, mitomycin C and thapsigargin
International
Hepatology ELSEJAER
International Hepatology Communications 2 (1994) 305-309
communications
Apoptosis and ~53 protein expression in human hepatoma cells induced by etoposide, mitomycin C and thapsigargin Yoshiyasu
Kaneko *, Ayumi Tsukamoto
First Department of Medicine. University of Tokyo, Faculty of Medicine, 7-3-Z Hongo, Bunkyo-ku, Tokyo 113, Japan
(Received 5 January 1994; accepted 16 February 1994)
Abstract We found that the anti-cancer drugs of mitomycin C and etoposide will induce chromatin decondensation in human PLCYPRFIS hepatoma cells, while the tumor promoter thapsigargin instead induces chromatin condensation. Both of these changes are subsequently followed by internucleosomal DNA cleavage and cell death characteristic to apoptosis. The p53 protein, which is known to induce apoptosis, increased in the anti-cancer drug-treated cells but not in thapsigargin-treated cells, thereby strongly suggesting that p53-dependent and -independent pathways linked to apoptosis exist in this hepatoma cell line. Key words: Centromere antibody; Anti-cancer drug; Thapsigargin; Tumor suppressor gene
1. Introduction Apoptosis is a nonpathological process necessary for organ formation and development [l], being postulated to be related with carcinogenesis since it is inhibited by phorbol esters that promote chemical carcinogenesis [2]. This process is also one of
the predominant pathways for anti-cancer drug action [3,4]. Although the exact mechanism is unknown, apoptosis is thought to be mediated via different pathways. In leukocytes, it can be induced artificially by glucocorticoids through Ca2+-dependent pathways, where calcium-dependent nucleases play a major role [5]. In some other kinds of cells, e.g., thymocytes, it has recently been suggested that p53-dependent and -independent pathways occur which induce apoptosis while bypassing calcium-dependent mechanisms [6,7]. In addition to apoptosis, non-apoptotic processes are believed to exist in programmed cell death [8]. As reported recently, since oncogene and anti-oncogene products and/or cell cycle *Corresponding author. 0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0928-4346(94)00013-U
306
Y. Kaneko, A. Tsukamotollnt. Hepatol. Commun. 2 (1994) 305-309
regulatory proteins are involved in processes linked to apoptotic cell death [1,4], investigations focused on these proteins are expected to further elucidate the mechanisms of carcinogenesis and apoptosis. This led to the present study, in which we examine the effects of anti-cancer drugs and a tumor promoter thapsigargin [9] on chromatin condensation, DNA cleavage, and p53 expression in human hepatoma cells. 2. Materials and methods PLC/PRF/S hepatoma cells (5 x 105) were grown in 5 ml of RPM11 640 medium plus 5% fetal calf serum with/without etoposide (1 ,&ml), mitomycin C (1 &ml), or thapsigargin (5 @ml). Nuclei were stained with 1 @ml acridine orange and examined using fluorescent microscopy [lo]. Centromeres were visualized by an indirect immunofluorescent antibody technique using anti-centromere antibody (Binding Site Ltd., Birmingham, UK) and fluorescent isothiocyanate (FITC)-conjugated anti-human IgG (Organon Technica Corp., PA, USA) [l 11. DNA fragmentation was analyzed by agarose gel electrophoresis [2,3]. Briefly, cells cultured with/without reagent were digested with proteinase K (250 ,@ml) and RNase A (20 pg/ml). DNA was isolated by the phenol method and separated by electrophoresis in 2% agarose gel containing 2 &ml of ethidium bromide [lo]. $X174 DNA/Hue111 fragments were used as a molecular weight marker. The nuclear p53 protein contents were analyzed by immunoblotting [l 11.We isolated nuclei from the cells and incubated them in a 0.35 M NaCl solution to extract non-histone proteins. Resulting nuclear proteins (10 pg) were run on a SDS-polyacrylamide gradient gel and blotted onto a nitrocellulose filter. p53 protein on the filter was visualized using specific monoclonal antibody (Oncogene Science, New York, USA) and a Blue Gene nonradioactive system (BRL Life Technologies, Inc., MD, USA). 3. Results The proliferation of PLC/PRF/S human hepatoma cells was almost completely inhibited when cultured with either 1 &ml etoposide, 1 pg/ml mitomycin C, or 5 r&ml thapsigargin. At the 4-day culture point, a significant increase in the cell number was not detected, with more than 95% of the cells being viable on a Trypan blue dye exclusion test. From 5 days however, cell viability gradually decreased and most cells were dead after 7 days. As shown in Fig. 1, the nuclei of cells cultured for 4 days with etoposide or mitomycin C became large and the centromeres were distributed within a wide nuclear space. In contrast, those of thapsigargin-treated cells became small and the centromeres existed in a narrow nuclear space. Of particular interest is that regardless of the changes in the nuclear structure, the number of centromeres per nucleus was not different among cells cultured with/without the different reagents (Fig. 1); hence indicating that the change in nuclear size reflects condensation and decondensation of chromatin.
Y. Kaneko, A. Tsukamotollnt. Hepatol. Commun. 2 (1994) 305-309
307
Fig. 1. The nuclear structure of PLC/PRF/S hepatoma cells (5 x 10’) after culturing with mitomycin C, etoposide, or thapsigargin. Cells were cultured for 4 days with mitomycin C (1 ,@ml), etoposide (1 &ml), or thapsigargin (5 &ml). Nuclei were stained with 1&ml acridine orange (A-D), while centromeres were visualized using anti-centromere antibody and FITC-conjugated second antibody (E-H). A and E, control; B en F, etoposide; C and G, mitomycin C; D and H, thapsigargin. ( x 1047).
Fig. 2 shows that culturing the cells with either mitomycin C or etoposide caused an internucleosomal DNA cleavage characteristic to apoptosis. Similar fragmentation is apparent in the DNA of free-floating cells cultured for 4 days with thapsigargin but not in corresponding monolayer cells. Since more than 95% of such free-floating cells exclude Trypan blue dye, this indicates that DNA cleavage in these cells is not due to reduced cell viability. M 1
2
3
M
4
5
Fig. 2. DNA fragmentation in treated PLCIPRFIS hepatoma cells. DNA from corresponding cells was electrophoresed after culturing for 4 days without (lane 1) or with 1 &ml etoposide (lane 2), 1 &ml mitomycin C (lane 3), or 5 ng/ml thapsigargin (lane 4, monolayer cells; lane 5, free-floating cells). Lane M, #Xl74 DNA/HaeIII digest.
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Y. Kaneko, A. Tsukamotolht.
km
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Hepatol. Commun. 2 (1994) 305-309
2
3
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2001007043 -
4-p53
2818 15 Fig. 3. The nuclear ~53 protein of PLCIPRF15 hepatoma cells after culturing with mitomycin C, etoposide, or thapsigargin. Cells were cultured for 4 days with/without reagents. Extracted nuclear proteins (10 fig) were subjected for immunoblot analysis using the anti-p53 antibody. Nuclear protein from control cells (lane 1), and cells treated with etoposide (lane 2), mitomycin C (lane 3), or thapsigargin (lane 4).
Immunoblot analysis of extracted nuclear proteins using anti-p53 antibody disclosed one discrete band at the expected molecular weight region (Fig. 3). The signal at 4 days was more intense in the etoposide- and mitomycin C-treated cells than in either the control or thapsigargin-treated ones (Fig. 3). Consequently, both mitomytin C and etoposide quantitatively increased the nuclear p53 protein content.
4. Discussion The nuclei of etoposide- and mitomycin C-treated PLC/PRF/S hepatoma cells became large, whereas the thapsigargin-treated cells became small, Since the centromeres were similarly distributed in respective wide and narrow nuclear spaces without a marked quantitative change, this suggests these agents induced decondensation or condensation of chromatin, respectively. Etoposide and mitomycin C are anti-cancer drugs known to cause DNA damage, which may be related to chromatin decondensation [12]. Thapsigargin in contrast has no direct effect on DNA and, therefore, the chromatin condensation observed in these treated cells is considered to be produced by changes in the intranuclear calcium level [9,13]. Such chromatin alterations were subsequently followed by internucleosomal DNA cleavage which is characteristic of apoptotic cell death (Fig. 2). The cells with fragmented DNA lost their membrane integrity and died; thereby indicating that apoptosis is responsible for inducing cell death caused by the employed reagents. This apoptotic process is hypothesized to be triggered by structural changes in chromatin which may increase the susceptibility of DNA to nucleases [5,6]. The p53 protein content increased in the etoposide- and mitomycin-treated cells (Fig. 3). DNA-damaging reagents have been recently reported to enhance p53 accumulation into the nuclei [14]. Moreover, increased amounts of p53 protein inhibit transition of cells from the Gl to S phase of the cell cycle and enable them to repair their damaged DNA [15]. It is also conceivable that the p53 protein which accumu-
Y. Kaneko, A. Tsukamotollnt. Hepatol. Commun. 2 (1994) 305-309
309
lates within the nuclei may directly trigger apoptosis via an unknown pathway [16]. Whether an increase in the ~53 protein directly alters the chromatin structure is of interest and must be investigated further. Because thapsigargin induced internucleosomal DNA cleavage and cell death without increasing the amount of this protein (Figs. 2 and 3), we suggest this hepatoma cell line has p53-independent pathways which are linked to apoptosis. As shown here, anti-cancer drugs and thapsigargin induce apoptosis by different molecular mechanisms, i.e., our results indicate the p53 protein plays an important role in anti-cancer drug-induced apoptosis, whereas some other growth- regulatory proteins are probably involved in thapsigargin-induced apoptosis. Further studies directed at apoptotic processes occurring in this cell line are expected to elucidate the mechanism of anti-cancer drug actions and carcinogenesis.
References [l] Williams GT, Smith CA. Molecular regulation of apoptosis: Genetic control on cell death. (Mini review) Cell 1993;74:777-779. [2] Tomei LD, Shapiro JP, Cope FO. Apoptosis in C3H/lOT1/2 mouse embryonic cells: Evidence for internucleosomal DNA modification in the absence of double-strand cleavage. Proc Nat1 Acad Sci USA 1993;90:853-857. [3] Nicotera P, Bellomo G, Orrenius S. Calcium-mediated mechanisms in chemically induced cell death. Annu Rev Pharmacol Toxic01 1992;32:449-470. [4] Williams GT. Programmed cell death: Apoptosis and oncogenesis. Cell 1991;65:1097-1098. [5] Alnemri ES, Litwack G. Activation of internucleosomal DNA cleavage on human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca*+-reguiring mechanism. J Biol Chem 1992;265:17323-17333. [6] Soignier MA, Hittlemen WN. Mitomycin-induced chromatid breaks in HeLa cells: A consequence of incomplete DNA replication. Cancer Res 1986;46:4032-4040. [7j Clark AR, Purdie CA, Harrison DJ, et al. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 1993;362:849-852. [8] Shwartz LM, Smith SW, Jones MEE, Osborne BA. Do all programmed cell deaths occur via apoptosis? Proc Nat1 Acad Sci USA 1993;90:98&984. [9] Thastrup 0, Cullen P, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca*+ stores by specific inhibition of endoplasmic reticulum Ca”-ATPase. Proc Nat1 Acad Sci USA 1990;87:24662470. [lo] Evans DL, Dive C. Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and nonproliferating thymocytes. Cancer Res 1993;53:2133-2139. [ll] Kaneko Y, Tsukamoto A, Kurokawa K. Vacuole formation and cytokeratin rearrangement of hepatoma cells induced by teleocidin are not associated with down regulation of protein kinase C. Cancer Res 1991;51:2677-2682. [12] Barry MA, Behnke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol 1990;40:2353-2362. [13] Tsukamoto A, Kaneko Y, Kurokawa K. Thapsigargin, an inhibitor of endoplasmic reticulum Ca2’ATPase, enhances c-fos expression but antagonizes vacuole formation of human hepatoma cells induced by teleocidin. Biochim Biophys Acta 1993;1177:31-36. [14] Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumor suppressor protein p53 by DNA-damaging agents, Oncogene 1993;8:307-318. [15] Levin AJ. The tumor suppressor genes. (Review) Annu Rev Biochem 1993;62:623-651. [16] Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A, Oren M. Wild-type ~53 induces apoptosis of myeloid leukemia cells that is inhibited by interleukin-6. Nature 1991;352:345-347.
Hepatology ELSEJAER
International Hepatology Communications 2 (1994) 305-309
communications
Apoptosis and ~53 protein expression in human hepatoma cells induced by etoposide, mitomycin C and thapsigargin Yoshiyasu
Kaneko *, Ayumi Tsukamoto
First Department of Medicine. University of Tokyo, Faculty of Medicine, 7-3-Z Hongo, Bunkyo-ku, Tokyo 113, Japan
(Received 5 January 1994; accepted 16 February 1994)
Abstract We found that the anti-cancer drugs of mitomycin C and etoposide will induce chromatin decondensation in human PLCYPRFIS hepatoma cells, while the tumor promoter thapsigargin instead induces chromatin condensation. Both of these changes are subsequently followed by internucleosomal DNA cleavage and cell death characteristic to apoptosis. The p53 protein, which is known to induce apoptosis, increased in the anti-cancer drug-treated cells but not in thapsigargin-treated cells, thereby strongly suggesting that p53-dependent and -independent pathways linked to apoptosis exist in this hepatoma cell line. Key words: Centromere antibody; Anti-cancer drug; Thapsigargin; Tumor suppressor gene
1. Introduction Apoptosis is a nonpathological process necessary for organ formation and development [l], being postulated to be related with carcinogenesis since it is inhibited by phorbol esters that promote chemical carcinogenesis [2]. This process is also one of
the predominant pathways for anti-cancer drug action [3,4]. Although the exact mechanism is unknown, apoptosis is thought to be mediated via different pathways. In leukocytes, it can be induced artificially by glucocorticoids through Ca2+-dependent pathways, where calcium-dependent nucleases play a major role [5]. In some other kinds of cells, e.g., thymocytes, it has recently been suggested that p53-dependent and -independent pathways occur which induce apoptosis while bypassing calcium-dependent mechanisms [6,7]. In addition to apoptosis, non-apoptotic processes are believed to exist in programmed cell death [8]. As reported recently, since oncogene and anti-oncogene products and/or cell cycle *Corresponding author. 0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0928-4346(94)00013-U
306
Y. Kaneko, A. Tsukamotollnt. Hepatol. Commun. 2 (1994) 305-309
regulatory proteins are involved in processes linked to apoptotic cell death [1,4], investigations focused on these proteins are expected to further elucidate the mechanisms of carcinogenesis and apoptosis. This led to the present study, in which we examine the effects of anti-cancer drugs and a tumor promoter thapsigargin [9] on chromatin condensation, DNA cleavage, and p53 expression in human hepatoma cells. 2. Materials and methods PLC/PRF/S hepatoma cells (5 x 105) were grown in 5 ml of RPM11 640 medium plus 5% fetal calf serum with/without etoposide (1 ,&ml), mitomycin C (1 &ml), or thapsigargin (5 @ml). Nuclei were stained with 1 @ml acridine orange and examined using fluorescent microscopy [lo]. Centromeres were visualized by an indirect immunofluorescent antibody technique using anti-centromere antibody (Binding Site Ltd., Birmingham, UK) and fluorescent isothiocyanate (FITC)-conjugated anti-human IgG (Organon Technica Corp., PA, USA) [l 11. DNA fragmentation was analyzed by agarose gel electrophoresis [2,3]. Briefly, cells cultured with/without reagent were digested with proteinase K (250 ,@ml) and RNase A (20 pg/ml). DNA was isolated by the phenol method and separated by electrophoresis in 2% agarose gel containing 2 &ml of ethidium bromide [lo]. $X174 DNA/Hue111 fragments were used as a molecular weight marker. The nuclear p53 protein contents were analyzed by immunoblotting [l 11.We isolated nuclei from the cells and incubated them in a 0.35 M NaCl solution to extract non-histone proteins. Resulting nuclear proteins (10 pg) were run on a SDS-polyacrylamide gradient gel and blotted onto a nitrocellulose filter. p53 protein on the filter was visualized using specific monoclonal antibody (Oncogene Science, New York, USA) and a Blue Gene nonradioactive system (BRL Life Technologies, Inc., MD, USA). 3. Results The proliferation of PLC/PRF/S human hepatoma cells was almost completely inhibited when cultured with either 1 &ml etoposide, 1 pg/ml mitomycin C, or 5 r&ml thapsigargin. At the 4-day culture point, a significant increase in the cell number was not detected, with more than 95% of the cells being viable on a Trypan blue dye exclusion test. From 5 days however, cell viability gradually decreased and most cells were dead after 7 days. As shown in Fig. 1, the nuclei of cells cultured for 4 days with etoposide or mitomycin C became large and the centromeres were distributed within a wide nuclear space. In contrast, those of thapsigargin-treated cells became small and the centromeres existed in a narrow nuclear space. Of particular interest is that regardless of the changes in the nuclear structure, the number of centromeres per nucleus was not different among cells cultured with/without the different reagents (Fig. 1); hence indicating that the change in nuclear size reflects condensation and decondensation of chromatin.
Y. Kaneko, A. Tsukamotollnt. Hepatol. Commun. 2 (1994) 305-309
307
Fig. 1. The nuclear structure of PLC/PRF/S hepatoma cells (5 x 10’) after culturing with mitomycin C, etoposide, or thapsigargin. Cells were cultured for 4 days with mitomycin C (1 ,@ml), etoposide (1 &ml), or thapsigargin (5 &ml). Nuclei were stained with 1&ml acridine orange (A-D), while centromeres were visualized using anti-centromere antibody and FITC-conjugated second antibody (E-H). A and E, control; B en F, etoposide; C and G, mitomycin C; D and H, thapsigargin. ( x 1047).
Fig. 2 shows that culturing the cells with either mitomycin C or etoposide caused an internucleosomal DNA cleavage characteristic to apoptosis. Similar fragmentation is apparent in the DNA of free-floating cells cultured for 4 days with thapsigargin but not in corresponding monolayer cells. Since more than 95% of such free-floating cells exclude Trypan blue dye, this indicates that DNA cleavage in these cells is not due to reduced cell viability. M 1
2
3
M
4
5
Fig. 2. DNA fragmentation in treated PLCIPRFIS hepatoma cells. DNA from corresponding cells was electrophoresed after culturing for 4 days without (lane 1) or with 1 &ml etoposide (lane 2), 1 &ml mitomycin C (lane 3), or 5 ng/ml thapsigargin (lane 4, monolayer cells; lane 5, free-floating cells). Lane M, #Xl74 DNA/HaeIII digest.
308
Y. Kaneko, A. Tsukamotolht.
km
1
Hepatol. Commun. 2 (1994) 305-309
2
3
4
2001007043 -
4-p53
2818 15 Fig. 3. The nuclear ~53 protein of PLCIPRF15 hepatoma cells after culturing with mitomycin C, etoposide, or thapsigargin. Cells were cultured for 4 days with/without reagents. Extracted nuclear proteins (10 fig) were subjected for immunoblot analysis using the anti-p53 antibody. Nuclear protein from control cells (lane 1), and cells treated with etoposide (lane 2), mitomycin C (lane 3), or thapsigargin (lane 4).
Immunoblot analysis of extracted nuclear proteins using anti-p53 antibody disclosed one discrete band at the expected molecular weight region (Fig. 3). The signal at 4 days was more intense in the etoposide- and mitomycin C-treated cells than in either the control or thapsigargin-treated ones (Fig. 3). Consequently, both mitomytin C and etoposide quantitatively increased the nuclear p53 protein content.
4. Discussion The nuclei of etoposide- and mitomycin C-treated PLC/PRF/S hepatoma cells became large, whereas the thapsigargin-treated cells became small, Since the centromeres were similarly distributed in respective wide and narrow nuclear spaces without a marked quantitative change, this suggests these agents induced decondensation or condensation of chromatin, respectively. Etoposide and mitomycin C are anti-cancer drugs known to cause DNA damage, which may be related to chromatin decondensation [12]. Thapsigargin in contrast has no direct effect on DNA and, therefore, the chromatin condensation observed in these treated cells is considered to be produced by changes in the intranuclear calcium level [9,13]. Such chromatin alterations were subsequently followed by internucleosomal DNA cleavage which is characteristic of apoptotic cell death (Fig. 2). The cells with fragmented DNA lost their membrane integrity and died; thereby indicating that apoptosis is responsible for inducing cell death caused by the employed reagents. This apoptotic process is hypothesized to be triggered by structural changes in chromatin which may increase the susceptibility of DNA to nucleases [5,6]. The p53 protein content increased in the etoposide- and mitomycin-treated cells (Fig. 3). DNA-damaging reagents have been recently reported to enhance p53 accumulation into the nuclei [14]. Moreover, increased amounts of p53 protein inhibit transition of cells from the Gl to S phase of the cell cycle and enable them to repair their damaged DNA [15]. It is also conceivable that the p53 protein which accumu-
Y. Kaneko, A. Tsukamotollnt. Hepatol. Commun. 2 (1994) 305-309
309
lates within the nuclei may directly trigger apoptosis via an unknown pathway [16]. Whether an increase in the ~53 protein directly alters the chromatin structure is of interest and must be investigated further. Because thapsigargin induced internucleosomal DNA cleavage and cell death without increasing the amount of this protein (Figs. 2 and 3), we suggest this hepatoma cell line has p53-independent pathways which are linked to apoptosis. As shown here, anti-cancer drugs and thapsigargin induce apoptosis by different molecular mechanisms, i.e., our results indicate the p53 protein plays an important role in anti-cancer drug-induced apoptosis, whereas some other growth- regulatory proteins are probably involved in thapsigargin-induced apoptosis. Further studies directed at apoptotic processes occurring in this cell line are expected to elucidate the mechanism of anti-cancer drug actions and carcinogenesis.
References [l] Williams GT, Smith CA. Molecular regulation of apoptosis: Genetic control on cell death. (Mini review) Cell 1993;74:777-779. [2] Tomei LD, Shapiro JP, Cope FO. Apoptosis in C3H/lOT1/2 mouse embryonic cells: Evidence for internucleosomal DNA modification in the absence of double-strand cleavage. Proc Nat1 Acad Sci USA 1993;90:853-857. [3] Nicotera P, Bellomo G, Orrenius S. Calcium-mediated mechanisms in chemically induced cell death. Annu Rev Pharmacol Toxic01 1992;32:449-470. [4] Williams GT. Programmed cell death: Apoptosis and oncogenesis. Cell 1991;65:1097-1098. [5] Alnemri ES, Litwack G. Activation of internucleosomal DNA cleavage on human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca*+-reguiring mechanism. J Biol Chem 1992;265:17323-17333. [6] Soignier MA, Hittlemen WN. Mitomycin-induced chromatid breaks in HeLa cells: A consequence of incomplete DNA replication. Cancer Res 1986;46:4032-4040. [7j Clark AR, Purdie CA, Harrison DJ, et al. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 1993;362:849-852. [8] Shwartz LM, Smith SW, Jones MEE, Osborne BA. Do all programmed cell deaths occur via apoptosis? Proc Nat1 Acad Sci USA 1993;90:98&984. [9] Thastrup 0, Cullen P, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca*+ stores by specific inhibition of endoplasmic reticulum Ca”-ATPase. Proc Nat1 Acad Sci USA 1990;87:24662470. [lo] Evans DL, Dive C. Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and nonproliferating thymocytes. Cancer Res 1993;53:2133-2139. [ll] Kaneko Y, Tsukamoto A, Kurokawa K. Vacuole formation and cytokeratin rearrangement of hepatoma cells induced by teleocidin are not associated with down regulation of protein kinase C. Cancer Res 1991;51:2677-2682. [12] Barry MA, Behnke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol 1990;40:2353-2362. [13] Tsukamoto A, Kaneko Y, Kurokawa K. Thapsigargin, an inhibitor of endoplasmic reticulum Ca2’ATPase, enhances c-fos expression but antagonizes vacuole formation of human hepatoma cells induced by teleocidin. Biochim Biophys Acta 1993;1177:31-36. [14] Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumor suppressor protein p53 by DNA-damaging agents, Oncogene 1993;8:307-318. [15] Levin AJ. The tumor suppressor genes. (Review) Annu Rev Biochem 1993;62:623-651. [16] Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A, Oren M. Wild-type ~53 induces apoptosis of myeloid leukemia cells that is inhibited by interleukin-6. Nature 1991;352:345-347.