Mutant p53 protein expression interferes with p53 ... - Nature

Oncogene (1998) 16, 3269 ± 3277  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc

Mutant p53 protein expression interferes with p53-independent apoptotic pathways Runzhao Li, Patrick D Sutphin, Dov Schwartz, Devorah Matas, Nava Almog, Roland Wolkowicz, Naomi Gold®nger, Huiping Pei, Miron Prokocimer and Varda Rotter Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 76100

Loss of normal p53 function was found frequently to interfere with response of cancer cells to conventional anticancer therapies. Since more than half of all human cancers possess p53 mutations, we decided to explore the involvement of mutant p53 in drug induced apoptosis. To further evaluate the relationship between the p53dependent and p53-independent apoptotic pathways, and to elucidate the function of mutant p53 in modulating these processes, we investigated the role of a p53 temperature-sensitive (ts) mutant in a number of apoptotic pathways induced by chemotherapeutic drugs that are currently used in cancer therapy. To that end, we studied the M1/2, myeloid p53 non-producer cells, and M1/2-derived temperature-sensitive mutant p53 expressing clones. Apoptosis caused by DNA damage induced with g-irradiation, doxorubicin or cisplatin, was enhanced in cells expressing wild type p53 as compared to that seen in parental p53 non-producer cells; mutant p53 expressing clones were found to be more resistant to apoptosis induced by these factors. Actinomycin D, a potent inhibitor of transcription, as well as a DNA damaging agent, abrogated the restraint apoptosis mediated by mutant p53. These observations suggest that while loss of wild type p53 function clearly reduces the rate of apoptosis, p53 mutations may result in a gain of function which signi®cantly interferes with chemotherapy induced apoptosis. Therefore, to achieve a successful cancer therapy, it is critical to consider the speci®c relationship between a given mutation in p53 and the chemotherapy selected. Keywords: p53-dependent; p53-independent; apoptosis; chemotherapeutic drugs

Introduction Immortalized tumor cells have lost their ability to undergo apoptosis, so it is conceivable that chemotherapeutic drugs should target the facilitation or restoration of apoptosis in cancer cells, rather than interfere with their enhanced proliferation (Cory et al., 1994; Hickman et al., 1994; Wyllie, 1994). Apoptosis is a multistage pathway regulated by a well characterized group of genes whose expression can be initiated through a variety of stimuli (Williams 1991; Oltvai and Korsmeyer, 1994; Wyllie, 1995).

Correspondence: V Rotter The ®rst two authors contributed equally to this paper Received 17 November 1997; revised 26 January 1998; accepted 26 January 1998

Wild type p53 was shown to play a central role in the induction of apoptosis (Yonish-Rouach et al., 1991; Lane, 1993; Lane et al., 1994; Wyllie et al., 1995; White, 1996). The observation that more than 50% of primary human tumors express mutant p53 proteins (Harris et al., 1986; Hollstein et al., 1991, 1994; Harris, 1993, 1996), coupled with the trend of most tumors to lose their apoptotic capacity, suggests that the two may be interconnected. It should be borne in mind however, that in addition to p53-dependent apoptotic pathways (Yonish-Rouach et al., 1991; Lu and Lane, 1993; Clarke et al., 1993, 1994; Lowe et al., 1993a,b; Strasser et al., 1994; Symonds et al., 1994), there are a number of p53-independent ones (Lowe et al., 1993b; Clarke et al., 1993; Berges et al., 1993; Kelley et al., 1994; Strasser et al., 1994; Zhuang et al., 1995) and it is possible that the interactions between these various pathways are important for the progression of apoptosis. Experiments comparing the apoptotic response of wild-type p53 to p53-null transformed embryonic ®broblasts, clearly demonstrated that p53 positively mediates the cytotoxicity of anticancer agents (Lowe et al., 1993a, 1994). However, there is evidence to support a less sensitive and an attenuated p53-independent apoptotic response to DNA damage (Strasser et al., 1994; Harrington et al., 1994; Lowe et al., 1994). Conclusions based on data obtained with in vitro experimental models suggest that expression of mutant p53 in cell lines hampers the onset of apoptosis (Lotem and Sachs, 1993, 1994) or cell dierentiation (Aloni-Grinstein et al., 1993, 1995; Soddu et al., 1996; Almog and Rotter, 1997). The possibility that mutant p53 acts by gain of function or by negative transdominant mechanisms has also been proposed. The notion that mutant p53 acts by gain of function is supported by the appearance of altered malignant phenotypes following the stable transfection of mutant p53 genes into p53-null cells. The increase in tumorigenicity following mutant p53 expression is particularly signi®cant. Furthermore, these studies suggest that dierent p53 mutations may vary in their ability to acquire these gained functions (Wolf et al., 1984; Halevy et al., 1990; Dittmer et al., 1993; Iwamoto et al., 1996). The idea that mutant p53 acts by a negative transdominant mechanism was mostly concluded from experiments in which concomitant expression of mutant p53 and wild type p53 rendered the latter inactive (Parada et al., 1984; Jenkins et al., 1984; Eliyahu et al., 1984; Levine, 1990). The signi®cance of the status of p53 with respect to its role in cancer therapy following drug administration is still ambiguous (Levine, 1997). It is not yet clear

Activity of mutant p53 in apoptosis R Li et al

p53ts-63

a

p53ts-61

whether the inability of primary human tumor cells to undergo apoptosis or terminal cell dierentiation is merely the result of loss of wild type p53 activity or the result of mutant gain of function. According to some reports, the level of drug-induced apoptosis increased when tumor cells expressed wild type p53, whereas mutant p53 producers were resistant. In other studies, no such correlation was described. Testicular cancer and Wilm's tumor retain an intact apoptotic response and are considered curable due to the fact that they rarely harbor mutations in the p53 gene (Masters et al., 1993; Oliver, 1996; Chresta et al., 1996). There are reports suggesting that chemosensitivity of breast cancer cells is either independent or dependent on the status of p53 (Makris et al., 1995; Aas et al., 1996). In addition, there are reports showing that in both breast (Allred et al., 1993) and in bladder cancers (Cote et al., 1997), chemotherapy favored cells expressing mutated p53 protein forms. In our previous studies we found that expression of mutant p53 in p53 non-producer cells protected them from apoptosis induced by growth-factor deprivation (Peled et al., 1996b). In contrast, wild type p53 expression induced an apoptotic response that cooperated with the p53-independent one. This presents direct evidence that certain p53 mutants may exert a direct oncogenic eect by interfering with apoptotic pathways. The goal of the present study was to further elucidate the eect of mutant p53 expression on p53independent apoptosis induced by a variety of DNAdamaging agents. We focused our study on drugs commonly used in the clinic, as well as g-irradiation. We found that expression of wild type p53 seems to augment the various p53-independent pathways studied. Mutant p53, which lost its apoptotic capacity, interrupted the p53-independent apoptotic pathways induced by growth factor removal, g-irradiation, doxorubicin and cisplatin. Interestingly, apoptosis induced by actinomycin D was not blocked by mutant p53, although as with the other drugs actinomycin D-

M1/2 p53ts-53

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b

induced apoptosis was enhanced by the expression of wild type p53. Results In our previous study we observed that while wild type p53 enhanced p53-independent apoptosis induced by growth-factor deprivation (removal of conditioned medium (CM)), expression of mutant p53 inhibited it (Peled et al., 1996b). In the present study, we have examined whether mutant p53 interferes with p53independent apoptosis induced by DNA damage. The experimental assay made use of stable clones derived from the M1/2, p53 non-producer cells, infected with a ts mutant p53 retroviral vector (pLXSNp53Val135). When cells are grown at 378C they predominantly express the mutant p53 protein conformation (Peled et al., 1996b). Shifting the temperature to 328C induces the expression of wild type p53 protein conformation (Gottlieb et al., 1994). Clones p53ts-53, p53ts-61 and p53ts-63 express high levels of the ts p53 mutant. Clone p53ts-53 expresses the least amount of p53 protein and clone p53ts-63 expresses the greatest (Peled et al., 1996b). Figure 1a depicts the relative amounts of p53 protein in the various clones studied, as estimated by Western blot analysis. Clone PLXSN generated by infection of the parental M1/2 cell line with the empty retroviral vector was used occasionally as a p53 non-producer control cell line (Peled et al., 1996b). Growth-factor deprivation induced apoptosis To con®rm our previous results, we examined whether expression of the wild type p53 conformation would enhance p53-independent apoptosis induced by CM removal, and if mutant p53 would interfere with the same apoptotic pathway. M1/2, p53 non-producers, and the ts mutant p53 producing clones p53ts-53 and p53ts-61, were examined

c

p53

Figure 1 Growth factor deprivation-induced apoptosis. (a) p53 protein levels in the various clones: 106 of cells of the dierent clones were lysed and analysed by Western blotting using the PAb-421 anti p53 monoclonal antibody. (b) Enhanced CM deprivation-induced apoptosis by wild type p53: M1/2, p53 non-producer cells, and clones p53ts-53 and p53ts-61, temperaturesensitive mutant p53 expressing cells derived from the M1/2 cell line, were shifted to 328C for 24 h with (striped column) and without CM (dotted column). Cell viability was then determined by FACS using the Propidium Iodide (PI) exclusion assay. Cell viability at 378C in the presence of CM (shaded column) is also included as a control. (c) Reduction in CM deprivation-induced apoptosis mediated by mutant p53: The kinetics of CM deprivation-induced apoptosis were examined at 378C. The PI exclusion based FACS assay was used to determine cell viability. The graph shows cell viability of the M1/2 (~), p53ts-53 (*), and p53ts-61 (~) cell lines as a percentage


for cell viability following CM deprivation. All cell lines were grown continuously at 378C in the presence of growth factor, prior to temperature shift or growthfactor deprivation. Figure 1b compares the cell viability of the M1/2 and the two temperature sensitive p53 clones at 378C and 328C in the presence of growth factor and at 328C without growth factor. Cell viability was determined using the Propidium Iodide (PI) exclusion assay. At 378C, in the presence of growth factor, all cells remain over 90% viable. Following a temperature shift from 378C to 328C, M1/2 cells remain over 90% viable while clones p53ts53 and p53ts-61 experience a signi®cant drop in cell viability induced by a p53-dependent apoptosis. Clones p53ts-53 and p53ts-61, following temperature shift to 328C and growth factor deprivation, also experience a drop in cell viability which appears to be signi®cantly greater than that caused by temperature shift alone. Under the same conditions M1/2 cells were not aected. Next we examined the eect of mutant p53 expression on growth-factor deprivation induced apoptosis. Figure 1c presents cell viability of parental M1/2, clone p53ts-53, and clone p53ts-61 cells grown at 378C following CM deprivation at progressive time points. M1/2 experienced a loss of cell viability, until ®nally, nearly no viable cells were detected. In contrast, clones p53ts-53 and p53ts-61, which express the mutant conformation of p53 at 378C, experienced little or no loss of cell viability following growth factor deprivation. The above data further con®rm that the concomitant induction of p53-independent apoptosis, via growth factor deprivation and p53-dependent apoptosis, cooperated to increase the rate of apoptosis relative to either apoptotic stimuli alone, while expression of mutant p53 interferes with p53-independent apoptosis induced through growth factor deprivation (Peled et al., 1996b).

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g-irradiation induced apoptosis To investigate the eects of wild type p53 expression on g-irradiation induced apoptosis, M1/2, clone p53ts53 and clone p53ts-61 cells, grown continuously at 378C, were temperature-shifted to 328C, to permit the expression of the wild type p53 conformation, with no treatment or following 800R g-irradiation. Figure 2a depicts the percent of cell survival through the FACSanalysis based PI exclusion assay. The temperatureshift alone resulted in the loss of cell viability in clone p53ts-53 and p53ts-61 that correlated with the levels of the expressed wild type p53 protein. M1/2 cells were unaected by the temperature-shift. Following 800R girradiation and incubation at 328C for 24 h, the M1/2 cells experienced a dramatic decrease in cell survival. girradiated, wild type p53 conformation expressing clones, clone p53ts-53 and clone p53ts-61, experienced a greater loss of cell viability relative to g-irradiated M1/2 p53 non-producer cells. This indicates that wild type p53 expression enhances the progress of girradiation induced apoptosis. To measure the eects of mutant p53 expression on g-irradiation, M1/2, clone p53ts-53, and clone p53ts-61 cells were examined for cell cycle distribution and cell death 72 h following 800R g-irradiation. Double staining of cells with PI and Hoescht-33342 con®rms that wild type p53 expression enhances g-irradiationinduced apoptosis, while mutant p53 interferes with it (Figure 2b). M1/2 cells experienced the greatest loss of cell viability. The mutant p53 expressing clones maintained a level of viability at least three times that of the M1/2 cells. Doxorubicin-induced apoptosis Following our experiments with g-irradiation, we examined if similar patterns of apoptosis would be exhibited by the two conformations of the temperature-

Figure 2 g-irradiation induced apoptosis. (a) Statistical representation of cell survival of the M1/2 (shaded column), p53ts-53 (striped column), and p53ts-61 (dotted column) cell lines as determined through the PI exclusion assay. Cells were temperatureshifted from 378C to 328C for 24 h with or without ®rst being g-irradiated at 800R. Cells were also maintained at 378C for 72 h following 800R g-irradiation. (b) Cell viability was determined through PI exclusion and DNA content through Hoescht-33342 staining. M1/2 cells, p53ts-53 and p53ts-61 were temperature-shifted from 378C to 328C for 24 h or g-irradiated at 800R (IR) followed by incubation at either 328C for 24 h, or 378C for 72 h. Ap-apoptotic cells; G1/G0; S; G2/M represents the distribution of cells at the various cell cycle phases: a, represents live cells; b, represents pre-apoptotic cells and c, represents apoptotic cells


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sensitive p53 protein in doxorubicin-induced apoptosis. This drug is the most widely used anthracycline antibiotic in cancer therapy. It intercalates into cellular DNA and aects many DNA-related functions, including DNA and RNA synthesis. Doxorubicin has also been shown to induce single and double strand DNA breaks that are believed to be mediated by the action of topoisomerase II or by the generation of free-radicals (Capranico and Zunino, 1992). Judged by cell morphology and DNA fragmentation it has been suggested that doxorubicin induces apoptosis (Ling et al., 1993; Skladanowski and Konopa, 1993; Zaleskis et al., 1994). To better specify the mode of cell death by Doxorubicin and its cell cycle pattern, we adopted the FACS-based Acrydine Orange (AO) DNA denaturability assay. This assay is based on the dierential denaturability of interphase, mitotic, and apoptotic DNA at low pH. Chromatin degradation speci®c for apoptosis renders DNA sensitive to denaturation (Darzynkiewicz et al., 1994). To that end, cells maintained at 378C were shifted to 328C for 24 h in the presence of increasing concentrations of doxorubicin. As can be seen in Figure 3a the wild type p53 expressing clones p53ts-53 and p53ts-63 underwent apoptosis without any treatment at 328C, while the M1/2 cells remained unaected by the temperature shift. In the presence of increasing concentrations of doxorubicin, low levels of apoptosis were observed in the M1/2 cell line, whereas there appeared to be a cooperative apoptotic eect in the wild type p53 expressing clones. This observed cooperation appeared to be more pronounced in the p53ts-63 clone. A likely explanation is that the p53ts-63 clone expresses higher basal levels of wild type p53 than clone p53ts-53 (see Figure 1a). Thus, the amount of apoptosis seems to be directly correlated to wild type p53 levels. At the restrictive temperature, 378C, mutant p53 appears to have an antagonistic eect on doxorubicin-

induced apoptosis. Figure 3b shows the percentage of apoptosis of cells treated at 378C for 24 h with increasing concentrations of doxorubicin. Clones p53ts-53 and p53ts-63, which express mutant p53, are less sensitive to the doxorubicin-induced apoptosis relative to the M1/2 parental cell line. These results support the conclusion that wild type p53 expression facilitated doxorubicin-induced apoptosis while mutant p53 expression antagonized it. It should be mentioned, however, that the sensitivity of M1/2 cells to the dierent drugs could be modi®ed following temperature shifting. Therefore, we consistently compared cell viability of the various clones at each given temperature. Cisplatin-induced apoptosis To further investigate the apoptotic patterns shown by the two conformations of the temperature-sensitive p53 mutant, we examined cisplatin-induced apoptosis with the AO assay. Like doxorubicin, cisplatin targets cellular DNA (Rosenberg, 1985). The type of DNA damage resultant of cisplatin treatment, however, diers from that caused by doxorubicin. Cisplatin damages DNA through cross-linking base pairs, most frequently via 1.2 interstrand adducts (Zamble and Lippard, 1995). It was suggested that the interstrand crosslinks are repaired via the nuclear excision repair pathway (Huang et al., 1994). Cells which are de®cient in this repair pathway, such as Xeroderma pigmentosum-derived ones are hypersensitive to cisplatin treatment (Dijit et al., 1988). Figure 4a depicts a typical apoptotic pattern of cells maintained at 378C and shifted to 328C for 24 h in the presence of increasing concentrations of cisplatin. The p53ts-53 and p53ts-63, wild type p53 expressing clones, underwent apoptosis without any treatment due to the high levels of wild type p53 expression. However, in the presence of the DNA damaging agent cisplatin, their levels of apoptosis were signi®cantly enhanced. The

Figure 3 Doxorubicin-induced apoptosis. A graphical representation of apoptosis as determined through the Acrydine Orange (AO) DNA-denaturability assay in the presence of increasing concentrations of doxorubicin. (a) M1/2 (&), p53ts-53 (~), and p53ts63 (&) cells were shifted from 378C to 328C, wild type p53 temperature, for 24 h with increasing concentrations of doxorubicin. (b) Quantitation of apoptosis following the treatment of the M1/2 (&), p53ts-53 (~), and p53ts-63 (&) cell lines with increasing concentrations of doxorubicin at 378C for 24 h


M1/2 cell line remained unaected by the temperatureshift and underwent apoptosis in a dosage dependent manner in the presence of increasing concentrations of cisplatin. However, the rate of apoptosis in M1/2 cells, did not reach those obtained following wild type p53 expression. Like doxorubicin, there appears to be a cooperative eect between the wild type p53-dependent apoptosis and the cisplatin-induced apoptosis. To investigate the eect of mutant p53 expression, M1/2, clone p53ts-53, and clone p53ts-63 cells maintained at 378C, were treated with increasing concentrations of cisplatin for 24 h. Figure 4b is a graphical representation of the percent of apoptotic cells assayed by AO. All cell types underwent apoptosis following treatment with cisplatin, with the M1/2 cells exhibiting the greatest sensitivity to the drug. The mutant p53 expressing clones, p53ts-53 and p53ts-63, experienced lower levels of apoptosis relative to M1/2 cells at the same cisplatin concentrations. These data are consistent with that of g-irradiation and doxorubicin-induced apoptosis. Mutant p53 expression interferes with DNA-damage induced apoptosis, caused by cisplatin, while wild type p53 cooperates with it. Actinomycin D-induced apoptosis Actinomycin D intercalates tightly and speci®cally into cellular DNA. The resultant drug-DNA complex then disrupts RNA chain elongation by DNA-dependent RNA polymerases, thereby inhibiting transcription and inducing the onset of apoptosis (Martin et al., 1990; Glynn et al., 1991; Lewis et al., 1995). Figure 5a shows the levels of apoptosis of M1/2 and clone p53ts-53, following a 24 h temperature shift from 378C to 328C, in the presence of increasing concentrations of actinomycin D. Cell viability was measured by PI exclusion assay. As observed previously, clones p53ts-53 underwent apoptosis

following temperature shift alone, while the M1/2 cell line remained unaected. Actinomycin D reduced cell viability in all of the cell lines examined. The reduction in cell viability in the wild type p53 expressing clone, however, by far exceeded the reduction in the level of cell viability in the p53 non-producing, M1/2 cells. To examine the eects of mutant p53 expression on actinomycin D-induced apoptosis, M1/2, clone p53ts53, and clone p53ts-61 cells were grown continuously at 378C, and treated with increasing concentrations of actinomycin D for 24 h at 378C. Apoptosis was measured by using the FACS-based AO DNAdenaturability assay. Percentage of apoptosis of the three cell lines examined is shown in Figure 5b. All cell lines underwent apoptosis following the 24 h treatment. Unlike the previous drugs tested, however, the apoptotic patterns of the three cell lines are indistinguishable regardless of p53 expression. Mutant p53 appears not to interfere with actinomycin D-induced apoptosis. This suggests that blockage of apoptosis by mutant p53 is dependent on a transcriptional controlled mechanism. In agreement with others, wild type p53dependent apoptosis is independent of transcriptional activity (Caelles et al., 1994). DNA fragmentation To con®rm our results obtained through FACSanalysis we evaluated DNA fragmentation using agarose gel electrophoresis. DNA fragmentation is a hallmark of apoptosis which has the appearance of a DNA ladder when resolved through gel electrophoresis. We examined DNA fragmentation following CM withdrawal, g-irradiation, doxorubicin treatment, cisplatin treatment and actinomycin D treatment of cells grown at 378C (Figure 6). Fortyeight and 72 h following conditioned medium with-

Figure 4 Cisplatin-induced apoptosis. Apoptosis is presented as the percentage of apoptotic cells in each cell line examined. Percentage of apoptotic cells was quantitated through the FACS-based AO DNA-denaturability assay. (a) M1/2 (&), p53ts-53 (~), and p53ts-63 (&) cells grown at 378C were temperature-shifted for 24 h in the presence of increasing concentrations of cisplatin. (b) Graphical representation of the percentage of apoptosis following treatment of M1/2 (&), p53ts-53 (~) and p53ts-63 (&) with increasing concentrations of cisplatin at 378C

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Figure 5 Eect of actinomycin D on wild type and mutant p53 induced apoptosis. (a) Cell viability (%) as determined through the FACS-based PI exclusion assay. M1/2 (shaded column) and p53ts-53 (striped column) were temperature-shifted from 378C to 328C in the presence of 0, 25, 50 and 75 ng/ml actinomycin D for 24 h. (b) M1/2 (&), p53ts-53 (~), and p53ts-61 (&) were treated for 24 h at 378C. Apoptosis was then quantitated using the AO DNA-denaturability assay

–CM M1/2 p53ts53 M 0 48 72 0 48 72

M1/2

+lR p53ts53

Doxorubicin M1/2 p53ts53

Cisplatin M1/2 PLXSN p53ts53

0 48 72 0 48 72 0 0.2 0.8 0 0.2 0.8 0 6 12 0 6 12 0 6 12

M1/2

Actinomycin D p53ts53 p53ts61

M 0 10 50 75 0 10 50 75 0 10 50 75

Figure 6 Eect of mutant p53 expression on p53-independent apoptosis: analysis by DNA fragmentation. 56105 cells were harvested and examined for DNA fragmentation through agarose gel electrophoresis following treatment with a variety of apoptotic stimuli at the restrictive temperature, 378C. DNA fragmentation was examined 48 and 72 h following either CM deprivation (7CM) or 800R g-irradiation. Cells were treated with cisplatin (5 mg/ml) for 0, 6 and 12 h. Cells were also examined 24 h following treatment with increasing concentrations of either doxorubicin (mg/ml) or actinomycin D (ng/ml). M1/2 and PLXSN are p53 nonproducer controls; p53ts-53 is a p53 producer derived cell line

drawal, agarose gel electrophoresis of genomic DNA revealed DNA ladders in the M1/2 cells, while clone p53ts-53 cells exhibited no DNA fragmentation. Similarly, 48 and 72 h following 800R g-irradiation, higher levels of DNA fragmentation were evident in the M1/2 cells, than in clone p53ts-53 cells. DNA fragmentation was also examined following treatment of M1/2, p53 non-producers and the mutant p53 expressing clones with increasing concentrations of either doxorubicin or actinomycin D for 24 h at 378C. Low levels of DNA ladders are present in the doxorubicin-treated M1/2 cells, while DNA fragmentation is almost absent in clone p53ts53. Treatment of p53 non-producer cell lines with cisplatin induced a typical DNA fragmented ladder. Indeed, M1/2 p53 non-producer cell line as well as the PLXSN cell line established by infection of the parental cell line with the `empty' retroviral vector (Peled et al., 1996b), gave similar apoptotic patterns which were already detected 6 h following treatment of cells with 5 mg/ml cisplatin. Treatment of p53ts-53

with cisplatin under the same conditions induced no augmented apoptosis. DNA fragmentation is clearly present following treatment of M1/2 cells with 75 ng/ml of actinomycin D. The actinomycin D-induced apoptotic DNA ladders seems as if they may actually appear at a lower concentration and at a greater extent in clone p53ts-53 and clone p53ts-61, than the M1/2 cells. These results con®rm the data collected through FACS-analysis, which suggested that mutant p53 expression interferes with apoptosis induced through conditioned medium withdrawal, g-irradiation, and doxorubicin treatment. On the other hand, mutant p53 does not seem to interfere with actinomycin D-induced apoptosis. Discussion Induction of programmed cell death or terminal cell dierentiation is the goal of successful cancer therapeutic treatment (Hickman et al., 1994). These


pathways can be induced by a variety of external signals such as growth factor deprivation, chemotherapeutic drugs, irradiation and others. The onset of the apoptotic pathway has been shown to be mediated by several independent routes, which all eventually converge into a speci®c death checkpoint leading to a single irreversible process (Williams, 1991; Oltvai and Korsmeyer, 1994; Symonds et al., 1994; Wyllie, 1995). The wild type p53, which is central to the onset of apoptosis, is lost in many tumor cells. Most primary tumor cells frequently express elevated levels of stable p53 mutant protein forms. Several studies have shown that constitutive expression of mutant p53 forms interferes with either the induction or the ongoing process of normal cell dierentiation (Aloni-Grinstein et al., 1993, 1995; Soddu et al., 1996) and apoptosis (Lotem and Sachs, 1993, 1994). In our present study we focused on the eects of the wild type and mutant conformations of the temperature-sensitive p53 protein in DNA-damage induced apoptosis mediated by g-irradiation and chemotherapeutic agents. We have recorded a synergistic eect on apoptosis following the concomitant induction of p53-dependent and p53-independent apoptosis induced by the DNA damage. Although M1/2 cells, p53 non-producers, still responded to DNA-damage through apoptosis, the apoptotic response was less sensitive as well as attenuated relative to cells expressing wild type p53. These data are consistent with those of others which demonstrate the existence of a p53-independent response to DNA damage (Liebermann et al., 1995) and that wild type p53 mediates the apoptotic response to anticancer drugs (Lowe et al., 1994). Indeed, it has been previously shown in a variety of cell lines, that mutant p53 confers resistance to DNA damaging agents (Lee and Bernstein, 1993; Eliopoulos et al., 1995; Hamada et al., 1996). p53-null tumors rarely occur in vivo. Therefore, we extended our studies to include mutant p53 expressing cells. Our data show that relative to p53 nonproducing cells, mutant p53 expressing cells are less sensitive to DNA damage-induced apoptosis. Since the cells used in this study dier only in their p53 status, it is reasonable to conclude that mutant p53 interferes with the p53-independent pathways of apoptosis. Also important, is the fact that these same cell lines readily underwent enhanced levels of apoptosis following DNA damage at the wild type p53 permissive temperature. This suggests that these cells maintain an intact p53-independent apoptotic response. Thus, any dierence in apoptosis observed at the restrictive temperature is resultant of mutant p53 expression. The mechanism by which mutant p53 interferes with p53-independent apoptosis still remains unclear. Our data suggest that mutant p53 may confer its antiapoptotic eect through a transcription-dependent manner. Results obtained following treatment of cells with Actinomycin D, a DNA damaging agent as well as a potent inhibitor of transcription, show that the protective eect may be abrogated through transcription inhibition. In agreement with others (Caelles et al., 1994), transcription inhibition mediated by actinomycin D did not interfere with the wild type p53 induced apoptosis. Moreover, actinomycin D induced apoptosis

seems to cooperate with the wild type p53 induced apoptosis. This con®rms the notion that the onset of p53-dependent apoptosis is a result of wild type p53 protein stabilization rather than enhancement of transcription, that is induced by DNA damage (Kastan et al., 1991, 1992; Di Leonardo et al., 1994). These data demonstrate that cancer therapies must not only contend with the loss of normal p53 function, but also with the gain of a mutant p53 anti-apoptotic function. Future studies should focus on clinically relevant p53 mutations and their involvement in p53independent apoptosis. Some mutations may be more potent inhibitors of apoptosis than others, while some p53 mutant types may remain susceptible to certain therapies. Importantly, not only have mutations in the p53 gene been found to dier in their ability to confer gained functions, but there is also evidence to suggest that loss of p53 function mutations may be uncoupled from gain of function mutations (Halevy et al., 1990). Anti-apoptotic function of mutant p53 may vary from one tumor type to another. Since some tumors expressing mutant p53 forms may remain refractory to conventional therapies, it is critical to develop therapeutic strategies to circumvent the possible anti-apoptotic function of mutant p53. Such strategies are already the focus of intense research. Recently, Roth et al. (1996) reported the use of retroviral mediated gene transfer of wild type p53 cDNA into patients with lung cancer who previously failed to respond to traditional therapies. Tumor regression was observed in three of nine patients and tumor growth stabilization was observed in another three (Roth et al., 1996). Also, a mutant adenovirus that selectively targets cells which lack wild type p53 function is currently under development (Bischo et al., 1996; Heise et al., 1997). Furthermore, a recent study has shown that introduction of a p53 C-terminal peptide can modulate mutant and wild type p53 function. The peptide induced tumor cell growth arrest and apoptosis (Selivanova et al., 1997). Taken together, the knowledge of whether mutant p53 can inhibit certain p53-independent apoptotic pathways induced by speci®c chemotherapeutic drugs could have major clinical rami®cations in the decision of which mode of therapy may be most suitable for each individual cancer expressing a particular type of p53.

Materials and methods Cell lines and culture medium The M1/2 cell line was derived from the M1-S6 parental murine myeloblastic cell line that was selected for its growth dependency on conditioned medium derived from the 14F1.1 bone marrow derived stromal cell line. The M1/2 cells were continuously grown in the presence of 10% conditioned medium (CM). CM was obtained from 14F1.1 cells that were grown to con¯uence and were collected every 3 ± 4 days, and maintained at 7208C prior to use (Peled et al., 1996a). Dierent CM batches exhibit variations in the content of growth factors and occasionally exhibit variations in their growth support capacity. To overcome this problem each experiment using an individual batch always included all controls and each point of the experiments was analysed as duplicates or triplicates.

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Drugs Actinomycin D (Sigma) and doxorubicin (Sigma) were prepared as a 500 mg/ml and 2 mg/ml stock solutions in water. Cisplatin (Abic) was prepared as a 1 mg/ml stock in water. Western blot analysis Anti-p53 monoclonal antibody; PAb-421 (Harlow et al, 1981) were puri®ed from ascitic ¯uids. For Western blot analysis, 106 cells were lysed in sample buer (140 mM Tris pH 6.8; 22.4% Glycerol; 6% SDS; 10% bmercaptoethanol and 0.02% Bromophenol Blue) and subjected to SDS ± PAGE. The proteins were detected using the protoblot Western blot Ap system (Promega). The nitrocellulose membranes were blocked with 10% dry skimmed milk in TBST (10 mM Tris-HCl pH: 8.0; 150 mM NaCl and 0.05% Tween-20) for 30 in. Incubation with the ®rst anti-p53 antibody was carried out for 30 min (ascitic ¯uid was diluted in TBST 1 : 100 or 1 : 200). Visualization of positive bands was performed according to the ECL protocol (Amersham). FACS analysis of apoptosis and cell cycle distribution Propidium Iodide (PI) in vivo labeling for viability and Hoechst-33342 labeling for DNA content: Cells, following dierent treatments at dierent time points, were incubated for 1 h with 5 mg/ml of Hoechst-33342 (Molecular Probes) dye which intercalates into DNA, and then for 3 min with 25 mg/ml PI (Sigma) which is incorporated into the ruptured membranes of dying cells (Belloc et al., 1994). The cells were analysed on a dual laser FACStar+cell sorter (Becton Dickinson Inc.). The UV laser excited Hoechst-33342 ¯uorescence was recorded on a FL/3 channel, and the PI ¯uorescence was recorded on the FL/ 2 channel. Using these two parameters the cells were sorted into two groups: Living-Low FL/2 ¯uorescence and DyingIntermediate FL/2 ¯uorescence. The sorting conditions were also selected for cells containing more than diploid DNA content according to FL/3 ¯uorescence. Quantitation of apoptosis using the AO DNA denaturability assay by FACS Cells were harvested and ®xed for at least 2 h in 80% ethanol ± 20% HBSS (Hanks Balanced Salt Solution) v/v,

at 7208C at 16106 cells/ml. The cells were washed once with HBSS and resuspended in HBSS with 0.25 mg/ml RNase A. After 1 h 15 min incubation at 378C, the cells were washed once and resuspended in HBSS at 26106 cells/ml. Cell suspensions (200 ml) were added to 0.5 ml of 0.1 M HCl. After 40 s incubation at RT, 2 ml of Acridine Orange (AO) (Molecular Probes) staining solution of pH 2.6, containing 90% v/v 0.1 M NaCitrate and 10% v/v 0.2 M Na2HP04 and 6 mg/ml AO, was added (Darzynkiewicz et al., 1994). Cells were analysed by the FACS using FACSort ¯ow cytometer and CellQuest list mode analysis software (BecktonDickinson) (Darzynkiewicz, 1994). The method is based on the metachromatic property of the AO dye. AO emits a green light upon the binding of double stranded nucleic acids and a red light when bound to single stranded nucleic acids. Detection of DNA Fragmentation (DNA ladder) 56105 cells were harvested, centrifuged and the medium was removed. The cells were suspended with 15 ml sample buer (10% glycerol, 10 mM Tris pH 8, 0.1 % (w/v) bromophenol blue)/RNase A (mixed at 1 : 1 volume). The cells were loaded into an agarose gel which contains two parts: The lower part (from the comb to the end of the gel) is a 2% agarose in TBE. The upper part (from the comb to the beginning of the gel) is a 1 % agarose, 2% SDS and 64 mg/ml proteinase K in TBE. The cells were electrophoresed for 10 h at 60 V at room temperature. The gel was stained with 2 mg/ml ethidium bromide in water for 1 h, and then destained with water (Eastman, 1995).

Acknowledgements The authors wish to thank David Wiseman for fruitful discussion and criticism. This work was supported in part by grants from the Leo and Julia Forchheimer Center for Molecular Genetics, and the Minerva Foundation. VR is the incumbent of the Norman and Helen Asher Professorial Chair in Cancer Research at the Weizmann Institute. Patrick D Sutphin (PDS) is a recipient of a Fulbright fellowship.

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