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Characterization of c-myc-Transformed Rat Fibroblasts Resistant to Apoptosis Induced by Growth Factor Deprivation
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
224, 52–62 (1996)
0110
Characterization of c-myc-Transformed Rat Fibroblasts Resistant to Apoptosis Induced by Growth Factor Deprivation SRIDEVI N. DHANARAJ, ALEXANDER M. MARCUS, REJU M. KORAH, KOICHI IWATA,
AND
MICHAEL B. SMALL1
Department of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical School, Newark, New Jersey, 07103-2714
apoptosis (reviewed in Refs. [6, 7]). This property of c-myc has been demonstrated in diverse cell types, including murine myeloid cells [8], primary and established rodent fibroblasts [9 – 11], T-cell hybridomas [12], and Burkitt lymphoma cells [13]. The precise mechanism by which c-myc contributes to the regulation of proliferation and apoptosis remains to be established. Myc, a nuclear-localized DNA-binding phosphoprotein, has been shown to function as a transcriptional activator in vitro [14–16], although its in vivo target genes remain largely unknown. Mutational analysis has revealed that the functional domains of c-Myc required for transformation are also necessary for its ability to mediate apoptosis [9]. Furthermore, dimerization of c-Myc with its DNA-binding partner Max is required for myc-mediated transformation and apoptosis [17, 18]. These results suggest that the transcriptional regulatory activities of c-myc are required for its apoptotic functions. It has been proposed that apoptosis may serve a protective role in preventing tumorigenicity elicited by deregulated Myc expression [19]. Tumor progression would therefore require a mechanism for abrogation of the myc-mediated cell death pathway. A number of possible secondary genetic events could account for such an outcome. First, an alteration in c-Myc (or Max) might impair the apoptotic function of c-Myc without affecting its mitogenic properties. Second, activation of a gene encoding an anti-apoptotic protein might be involved. In this regard, it has been shown that overexpression of human Bcl-2 [10, 20, 21], Bcl-xL [22], or the adenovirus E1B 19K protein [22] is capable of blocking myc-mediated apoptosis without affecting the transformed phenotype. In addition, Myc and Bcl-2 appear to act synergistically in several tumor cell systems [23, 24]. Third, it has been demonstrated that serum cytokines, in particular the insulin-like growth factors, act as survival signals to protect cells against myc-mediated apoptosis [25]. A mutation leading to constitutive activation of the signal transduction pathways through which these factors operate could therefore lead to resistance to apoptosis induced by growth factor deprivation. Finally, apoptosis could be blocked by inactivation of an integral component in the cell death machinery. In this study, we have sought to further examine
Under appropriate conditions (e.g., growth factor withdrawal), the deregulated expression of c-myc in rodent fibroblasts leads to substantial cell death due to apoptosis. To better understand this process, we selected for c-myc-transformed Rat1A fibroblasts that were resistant to growth factor deprivation-induced cell death. One clonal isolate exhibited prolonged survival in serum-free medium and displayed reduced levels of apoptosis-related DNA fragmentation. These cells were also resistant to induction of apoptosis by the protein kinase inhibitor staurosporine. They retained a transformed cell phenotype and expressed the proviral human c-myc allele in an unaltered fashion, strongly indicating that the mutation of a cellular gene other than c-myc accounts for the apoptosis-resistant phenotype. The results of somatic cell hybrid analysis of this cell line are consistent with a recessive mutation. Our findings suggest a novel mechanism for abrogation of apoptosis in neoplastic cells and provide a model system for the study of its role in tumorigenesis and resistance to antineoplastic therapy. q 1996 Academic Press, Inc.
INTRODUCTION
The c-myc proto-oncogene has long been recognized as an important regulator of normal and malignant cell growth (reviewed in Refs. [1 – 3]). Expression of cmyc serves as an essential signal for entry and progression through the cell cycle and as a modulator of cell differentiation. Deregulation of c-myc is strongly implicated in the pathogenesis of a variety of human tumors, and its in vitro transforming activity has been clearly demonstrated [4, 5]. Most recently, it has become clear that the c-Myc oncoprotein has pleiotropic functions that act in opposition to its role as a mediator of cell proliferation. Specifically, it has been shown that, under appropriate conditions (e.g., growth factor deprivation), the deregulated expression of c-myc can promote cell death by a process attributable to 1
To whom correspondence and reprint requests should be addressed. Fax: (201) 982-3644. 52
0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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the mechanisms involved in myc-mediated apoptosis through the selection of apoptosis-resistant mutants of c-myc-transformed Rat1A fibroblasts. A clonal isolate exhibited resistance to cell death induced by both serum withdrawal and the general protein kinase inhibitor staurosporine, while retaining the transformed cell phenotype. On the basis of several criteria, we believe that a recessive-acting mutation in a cellular gene other than c-myc itself is responsible for the apoptosisresistant phenotype. Our findings suggest a novel mechanism for abrogation of apoptosis in neoplastic cells and provide a model system for the study of its role in tumorigenesis and resistance to antineoplastic therapy. MATERIALS AND METHODS Cell lines and cell culture. The parental transformed cell line used in these studies, designated RMN8, was generated by infection of Rat1A cells with a retroviral stock derived by transfection of C2 packaging cells [26] with the retroviral vector pMV6myc [21]. pMV6myc expresses a human c-myc cDNA from a murine leukemia viral long terminal repeat (LTR) and the bacterial gene that confers resistance to the antibiotic G418 under the control of an internal thymidine kinase promoter. RMN8 cells were initially selected in complete medium (see below) containing 800 mg/ml G418 and then recloned in 0.35% agarose (SeaPlaque). The myc/bcl2 polyclonal cell line was described previously [21]. For cell fusion studies, hygromycin B-resistant derivatives of nontransformed (Rat1A/hyg) and cmyc-transformed (myc/hyg) Rat1A cells were generated by infection with pMV12 and pMV12myc retroviruses, respectively [21]. Cell lines were maintained in Dulbecco’s modified Eagle’s medium/ F-10 (1:1) containing 10% newborn calf serum (complete medium) at 377C in 7.5% CO2 . Anchorage-independent growth was measured as described [4]. Two hundred cells were resuspended in agarose (containing complete medium) and overlaid into triplicate 60-mm dishes that were visually inspected for colony formation after incubation at 377C for 14 to 21 days. Isolation of apoptosis-resistant cells. RMN8 cells were initially seeded in fifty 100-mm dishes at 105 cells per dish in complete medium. The following day, cells were washed with phosphate-buffered saline (PBS), refed with serum-free medium, and incubated at 377C for 6 days. These conditions have previously been determined to result in virtually complete death of these cells by apoptosis. Plates were then refed with complete medium to allow viable cells to recover and form colonies. One week later, 35 surviving colonies were isolated and expanded into mass culture in medium containing 400 mg/ ml G418, indicative of continued expression of the integrated c-myc provirus. All 35 colonies were then reexamined for enhanced viability in serum-free media, as assessed qualitatively by increase in cell number or decrease in floating (detached) cells. Determination of cell viability and apoptosis. For viability and apoptosis assays, cells were seeded in complete medium at 5 1 104 cells per 100-mm dish. Twenty-four hours later, duplicate dishes were washed with PBS, refed with complete or serum-free medium, with and without 22 or 50 nM staurosporine (Calbiochem), and cultured for a period of 72 h. Detached and adherent cells (harvested by trypsinization) were resuspended in PBS and counted in a Royco cell counter (total cell counts). Total viable cell number was determined by trypan blue exclusion at various times after induction of cell death, as described [21]. For quantitation of apoptosis, detached and adherent cells were pooled, washed in PBS, and collected by centrifugation at 1200g for 10 min. Cells were resuspended in 1:10 volume of cold fixing solution (PBS with 5% NCS and 1% formaldehyde). Ten microliters of fixed
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cell suspension was placed on an acridine orange-coated slide, sealed with a coverslip, and observed by fluorescent microscopy [27]. The average number of apoptotic cells (i.e., those displaying chromatin condensation or formation of apoptotic bodies) was determined by counting 200 cells (in each of three separate fields) on triplicate slides. The percentage of apoptotic cells was calculated by dividing the number of apoptotic cells by total cell number and multiplying by 100. For DNA fragmentation analysis, total DNA was isolated from subconfluent cell populations by lysis in buffer containing 0.5% SDS, 10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 50 mM EDTA, and 500 mg/ml proteinase K followed by phenol–chloroform extraction and ethanol precipitation. One hundred nanograms of each DNA sample was end-labeled with [a-32P]dideoxyATP using terminal transferase [28]. Following termination of the labeling reaction, DNA was ethanol precipitated, collected by centrifugation, and resuspended in 20 ml of 10 mM Tris–HCl, 1 mM EDTA, pH 7.5. Thirty-five nanograms of each labeled DNA was electrophoresed on a 1.8% agarose gel in Tris–acetate–EDTA buffer at 50 V for 6 h. The gel was fixed in 10% trichloroacetic acid for 30 min, rinsed in distilled water, vacuumdried, and exposed to autoradiographic film overnight. Fluorimetric quantitation of low-molecular-weight DNA, isolated by detergent lysis and differential centrifugation, was performed by an adaptation of the method as described in Ref. [29]. Flow cytometric analysis. Cells were harvested by trypsinization and washed three times in ice-cold PBS (Mg2/-Ca2/-free). Cells in 0.9 ml of PBS were passed through a 25-gauge needle and fixed by dropwise addition into 2.1 ml of ice-cold 100% ethanol. Approximately 106 fixed cells were resuspended in 0.9 ml PBS containing 7.5 mg/ml DNase-free RNase A. Cells were incubated at 377C for 1 h and stained with 50 mg/ml propidium iodide. Cell cycle distributions were determined by analysis of 104 cells using the CellFIT program on a FACScan flow cytometer (Becton-Dickinson). Proviral rescue by superinfection with murine leukemia virus. Cells were assayed for the presence of a functional human c-myc gene by superinfection with Moloney murine leukemia virus (MLV) and generation of pseudotype retrovirus [30]. Cells were seeded at 5 1 105 per 100-mm dish, infected the following day with 2 ml of MLV-containing medium (a gift of M. Roth, UMDNJ-Robert Wood Johnson Medical School) supplemented with 8 mg/ml Polybrene, and then refed 4 h later with complete medium. Medium was collected from superinfected cells 48 h postinfection and passed through a 0.45-mm Millipore filter, and 2 ml of this medium was used to infect Rat1A cells, seeded at 5 1 105 per 100-mm dish, as described above. Forty-eight hours postinfection, infected Rat1A cells were trypsinized and reseeded into complete medium containing 400 mg/ml G418. After 7 to 10 days, dishes were examined for the presence of G418resistant colonies. Approximately 100 colonies from each of two separate dishes were pooled to generate independent cell populations (e.g., AR27-RP1 and AR27-RP2), and viability and anchorage dependence assays were performed on each pool as described previously. Preparation of nuclear and whole cell extracts. Nuclear extracts were prepared as described [31]. Protein concentrations were determined by the Bradford assay and extracts were stored at 0707C. Whole cell extracts (for p53 analyses) were prepared by lysis of one 100-mm subconfluent dish in 1 ml of lysis buffer (25 mM Tris– HCl, pH 7.5, 100 mM NaCl, 2% NP-40, 0.2% SDS, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mg/ ml aprotinin, and 50 mM leupeptin). Lysates were incubated for 30 min at 47C and then centrifuged (12,000g) for 15 min at 47C. The supernatant was collected and used for p53 immunoprecipitation. Analysis of c-Myc and p53 protein. One hundred micrograms of protein from nuclear extracts in sample buffer containing 33% glycerol, 0.3 M DTT, 6.7% SDS, and 0.01% bromophenol blue was electrophoresed on a 10% SDS–polyacrylamide gel at 30 V for 18 h. Separated proteins were transferred to 0.2-mm nitrocellulose (Schleicher & Schuell) using a Hoeffer transfer apparatus. Nitrocellulose filters were blocked for 1 h with 5% (w/v) nonfat dry milk in
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buffer containing 10 mM Tris–HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20 (TBST), and then incubated with mouse monoclonal antibody 9E10 (Oncogene Science), diluted 1:20, for detection of c-Myc protein. Filters were washed in TBST and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat antimouse antibody. Filters were developed in ECL detection reagent (Amersham) for 1 min and exposed to X-ray film for 10 s. For determination of p53 status, whole cell extracts (300 ml) were immunoprecipitated overnight at 47C with 1 mg of each of the following anti-p53 monoclonal antibodies (Oncogene Science): wild-type specific Pab 246, mutant-specific Pab 240, or pan-specific Pab 421 (which detects both wild-type and mutant p53). Protein G-Plus agarose beads (Oncogene Science) were added and incubated for 30 min at 47C as described [32]. The immunoprecipitates were washed extensively in lysis buffer, boiled in SDS sample buffer, and then resolved by SDS–polyacrylamide gel electrophoresis and immunoblotting as described above. p53 was detected using Pab 421 as the primary antibody and an anti-mouse k light chain peroxidase-conjugated secondary antibody (Zymed), followed by ECL detection. Somatic cell hybrid analysis. AR27 (2.5 1 105) cells were fused to Rat1A/hyg or myc/hyg (2.5 1 106) cells by treatment with 50% w/v polyethylene glycol (PEG) 1500 for 1 min [33]. Cells were then reseeded into complete medium containing G418 (400 mg/ml) and hygromycin B (200 mg/ml). For production of self-hybrids, AR27 derivatives resistant to hygromycin B or expressing the bacterial gene gpt were generated by calcium phosphate-mediated transfection with the plasmids pSV2gpt [34] or puc/hyg [4], respectively. Following PEG fusion, AR27 self-hybrids were selected in complete medium containing hygromycin B (200 mg/ml), 25 mg/ml mycophenolic acid, and 75 mg/ml xanthine. Hybrid colonies were isolated, expanded into mass culture, and assessed for serum deprivation-induced apoptosis by acridine orange staining as described above.
RESULTS
Isolation of Apoptosis-Resistant Cell Line AR27 Derivatives of the established rodent fibroblast cell line Rat1A transformed by human c-myc undergo apoptosis when deprived of serum growth factors in vitro [9, 21]. To further understand the mechanisms involved in myc-mediated apoptosis, we undertook to isolate apoptosis-resistant mutants of myc-transformed Rat1A by selection of cells capable of prolonged survival in serum-free medium. This strategy would not be expected to have a bias for or against retention of the transformed phenotype. The parental cell line used in these studies, designated RMN8, expresses a human c-myc allele introduced by infection of Rat1A cells with the retrovirus pMV6myc, which also encodes the bacterial gene conferring resistance to the antibiotic G418 (see Materials and Methods). RMN8 cells, selected initially for G418 resistance, display a transformed cell morphology and are able to form colonies in soft agarose. Southern analysis (data not shown) demonstrated that these cells harbor a single pMV6myc provirus. A total of 5 1 106 RMN8 cells, initially seeded at 105 cells per 100-mm dish in complete medium, were washed with PBS, refed with serum-free medium, and incubated at 377C for 6 days. Under these conditions, the vast majority of cells underwent programmed cell death and detached from the dish, a phenotype associ-
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ated with apoptosis in these cells [9]. Plates were then refed with complete medium to allow surviving cells to recover and form colonies. One week later, 35 colonies were isolated and expanded into mass culture in medium containing the antibiotic G418, indicative of continued expression of the integrated c-myc provirus. All 35 colonies were then reexamined individually for viability in serum-free medium. One clone, designated AR27, displayed prolonged survival in serum-free medium relative to the parental RMN8 cell line, as assessed qualitatively by either an increase in cell number or a decrease in detached cells. Unlike the other clones tested, this phenotype was stable upon continued passage in culture and was independent of cell density (compare Figs. 1A and B). Furthermore, AR27 cells exhibit a transformed cell morphology in complete medium indistinguishable from that of parental RMN8 cells, although they appear somewhat flatter and less refractile in serum-free medium (see Fig. 1A). Maintenance of the transformed cell phenotype of AR27 was also confirmed by assay for growth in soft agarose. As shown in Table 1, AR27 cells form anchorage-independent colonies (as well as colonies in monolayer culture) with efficiencies comparable to the RMN8 cells. Cell cycle analysis by flow cytometry (see Table 2) confirmed that AR27 displays a cycling cell distribution after 24 h in serum-free medium, indistinguishable from parental RMN8 cells. We determined total viable cell counts (using trypan blue exclusion) of AR27 in complete and serum-free media, in comparison to the parental RMN8 cell line and another clone (AR35) which had emerged from the initial selection. Figure 2A shows that the growth rates in complete medium for AR27 and AR35 are similar to that of RMN8. In serum-free medium (Fig. 2B), AR27 cells continued to proliferate, albeit at a slower rate than in complete medium, and retained nearly 100% viability following 72 h of serum withdrawal. In contrast, parental RMN8 and AR35 cells exhibited only a slight increase in cell number over the same time period. These results reflect the documented ability of deregulated c-myc expression to promote cell proliferation in the absence of serum growth factors [35], offset in the case of RMN8 and AR35 cells by a 70% decrease in cell viability. Thus, the greater net increase in cell number of AR27 in serum-free medium, relative to that of RMN8 and AR35, is attributable to enhanced viability, rather than increased proliferative ability. Resistance of AR27 to Serum Deprivation-Induced Apoptosis We next sought to determine whether the increased viability in serum-free medium displayed by AR27 was due to resistance to apoptotic cell death induced by growth factor withdrawal. The degradation of nuclear DNA into fragments of nucleosome length units (or
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FIG. 1. Cell morphology in complete and serum-free medium. Parental RMN8 and apoptosis-resistant AR27 cells were seeded at 1 1 105 (A) or 1 1 104 (B) per 100-mm dish. Cells were photographed after 5 days of growth factor deprivation. (Original magnification, 1100.)
multiples thereof) is widely regarded as a reliable biomarker for apoptosis (reviewed in Ref. [6]). We therefore analyzed AR27 cells for evidence of apoptosis-specific DNA fragmentation (i.e., nucleosome ladders) upon culture in serum-free medium. For comparative purposes, we included in our analysis nontransformed Rat1A and parental RMN8 cells, as well as a myctransformed Rat1A derivative that overexpresses the human bcl2 gene (myc/bcl2) and is resistant to apoptosis [21]. Total DNA was isolated from both adherent and detached cells grown in either complete or serum-free medium for 48 h. Equal amounts of DNA from each cell line under both growth conditions, radiolabeled by terminal transferase, were resolved by agar-
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ose gel electrophoresis. As shown in Fig. 3, AR27 exhibits significantly reduced levels of nucleosome-unitlength fragments in serum-free medium (lane 6), as compared to parental RMN8 cells grown under the same conditions (lane 4). The level of DNA fragmentation in AR27 appears to be even lower than that observed in myc/bcl2 cells (lane 8). Comparable results were obtained by fluorimetric quantitation of Hoechst 33258-stained low-molecular-weight DNA isolated by differential centrifugation of detergent-lysed cells (data not shown). We also examined the same cell lines at various times after serum deprivation for morphological changes characteristic of apoptosis using fluorescence
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TABLE 1 Efficiency of Colony Formationa Cell line
Agarose
Monolayer
Rat1A RMN8 AR27 RMN8-RP1 RMN8-RP2 AR27-RP1 AR27-RP2
õ1 26 22 28 25 19 21
40 28 26 38 35 26 31
a Efficiency of colony formation as percentage of input cells; 200 cells were seeded in agarose or monolayer in duplicate dishes as described under Materials and Methods.
microscopy of acridine orange-stained cells. This method of analysis allowed us to more precisely quantify the percentage of apoptotic cells at specific time points. As shown in Fig. 4, AR27 cells are as resistant as myc/bcl2 cells, even 72 h after withdrawal of growth factors. In contrast, greater than 70% of the parental RMN8 cells exhibit apoptotic changes by the same time point. Thus, all three methods used to measure apoptosis, in conjunction with cell proliferation and viability assays, have yielded consistent data that AR27 cells are resistant to serum deprivation-induced cell death. Retention of Functional c-myc Expression in AR27 As described above, AR27 cells are resistant to apoptosis induced by growth factor withdrawal, yet continue to manifest a transformed cell phenotype characteristic of the RMN8 cell line from which they were derived. The persistence of the transformed phenotype would suggest that these cells continue to express functional c-myc RNA and protein. Southern analysis (data not shown) revealed no apparent alteration in the structure of the integrated pMV6myc proviral DNA. However, it has previously been shown that transformation and apoptosis mediated by c-myc are dosage-dependent phenomena [4, 9]. It was conceivable that reduced c-myc expression in AR27 might account for resistance to myc-mediated apoptosis, without affecting the transformed phenotype. To address this possibility, we compared the levels of human c-Myc protein in nuclear extracts from parental RMN8 and AR27 cells by Western blot analysis. As shown in Fig. 5, AR27 (lane 3) expresses steady state levels of c-Myc protein equivalent to those of RMN8 (lane 2). Northern blot and RNase protection analysis of AR27 also revealed no alteration in c-myc expression at the transcriptional level (data not shown). Similar results were found in cells cultured in complete medium as well as in serumfree medium, consistent with the absence of a serum responsive element in the retroviral promoter driving c-myc expression.
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To assess the functional status of the human c-myc allele in AR27, we superinfected these cells, in parallel with the parental RMN8 cell line, with Moloney murine leukemia virus (MLV), giving rise to cells producing a mixed population of wild-type MLV and pseudotype retrovirus [30] containing the human c-myc proviral transcript. Tissue culture supernatants from the MLVinfected cell lines were used to infect nontransformed Rat1A cells, which were then selected by colony formation in medium containing the antibiotic G418. G418resistant colonies were pooled, expanded into mass culture, and examined for phenotypic transformation as well as for their ability to undergo apoptosis upon serum withdrawal. Pooled cell populations (AR27-RP1 and RP2) derived by infection with rescued virus from AR27 displayed a transformed cell morphology and an anchorage-independent phenotype (see Table 1) no different from cells generated by infection with rescued virus from RMN8 (RMN8-RP1 and RP2). As shown in Fig. 6, all pooled rescue cell lines exhibited a loss of cell viability upon serum withdrawal, as measured by trypan blue exclusion, indistinguishable from one another in both magnitude as well as time course. Microscopic examination of these cells (data not shown) confirmed that cell death was due to apoptosis. On the basis of these findings, we conclude that the human c-myc allele resident in AR27 is unaltered in expression and function. Thus, the apoptosis-resistant phenotype is consistent with a mutation in a cellular gene other than c-myc itself. These results also provide evidence for independent components in the pathways by which c-myc mediates transformation and apoptosis (see Discussion). Cross-Resistance of AR27 to Induction of Apoptosis by Staurosporine We considered the possibility that AR27, selected for resistance to serum deprivation-induced apoptosis, might also be resistant to induction of programmed cell death by other agents. Cross resistance to multiple
TABLE 2 Cell Cycle Analysis in Complete and Serum-Free Medium % of cells (mean { SD) in: Cell line
Serum
RAT1A RMN8 AR27 RAT1A RMN8 AR27
/ / / 0 0 0
G0/G1 34.8 38.6 39.9 78.2 55.9 57.8
{ { { { { {
0.1 1.7 0.1 0.4 0.1 0.0
S 51.4 43.6 44.5 15.0 33.3 30.6
{ { { { { {
G2/M 0.0 3.7 0.1 0.0 0.0 0.1
13.8 17.8 15.8 6.8 10.8 11.7
{ { { { { {
0.1 2.1 0.1 0.4 0.0 0.1
Note. SD, standard deviation; data represent two independent experiments.
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FIG. 2. Growth curves of parental and apoptosis-resistant cells. Cells seeded at 5 1 104 per 100-mm dish were grown in complete (A) or serum-free (B) medium. Total viable cell counts were determined as described under Materials and Methods. Error bars represent standard deviation from the mean.
inducers of apoptosis might suggest that the mutation responsible for apoptosis resistance directly involves a component of the cell death machinery. We chose to examine the response of AR27 to treatment with the general protein kinase inhibitor staurosporine, which has been shown to induce apoptosis in a wide variety of cell types [36]. AR27 cells, along with RMN8 and myc/bcl2 cells, were cultured in complete medium in
the absence and presence of 22 nM (low) and 50 nM (high) staurosporine. Apoptosis, as quantitated by acridine orange staining, was measured after 48 h of treatment under these conditions. As shown in Fig. 7, parental RMN8 cells display significant cell death at both concentrations tested. In contrast, AR27 cells are markedly resistant to staurosporine-induced cell death at 22 nM, although they display only partial resistance at the higher concentration. In comparison, myc/bcl2 cells are protected at both low and high staurosporine levels. These results suggest that the apoptosis-resistant phenotype of AR27 may involve a mutation in a component of the cell death pathway common to induction by both growth factor withdrawal and staurosporine. Measurements of cell viability, as determined by trypan blue exclusion, parallel the results we have obtained by acridine orange staining (data not shown). Genetic Analysis of the Apoptosis-Resistant Phenotype of AR27
FIG. 3. Analysis of DNA fragmentation. Total DNA was isolated from adherent and detached cells, radiolabeled with terminal transferase, and resolved by electrophoresis on 1.8% agarose gel. Rat1A (lanes 1 and 2), RMN8 (lanes 3 and 4), AR27 (lanes 5 and 6), and Myc/Bcl2 (lanes 7 and 8) cells were grown for 48 h in complete (lanes 1, 3, 5, and 7) or serum-free (lanes 2, 4, 6, and 8) medium.
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In order to determine the genetic nature (i.e., dominance or recessiveness) of the apoptosis-resistant phenotype of AR27, we generated somatic cell hybrids between AR27, which is resistant to the antibiotic G418, and both nontransformed and c-myc-transformed Rat1A cell lines expressing the bacterial gene encoding resistance to the antibiotic hygromycin B (see Materials and Methods). Hybrid cells were selected by colony formation in complete medium supplemented with G418 and hygromycin B, conditions shown to kill the unhybridized parental cells. Individual colonies were tested for survival in serum-free medium. Qualitative observations suggested that the apoptosis-sensitive phenotype was restored in the majority of hybrids examined. We then used fluorescence microscopy of acridine orange-stained cells to quantitate the degree of
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FIG. 4. Quantitation of apoptosis in parental and apoptosis-resistant cells. Adherent and detached cells were stained with acridine orange following culture in complete or serum-free medium for 0, 12, 24, 48, and 72 h and observed by fluorescent microscopy. Percentage apoptosis was calculated as described under Materials and Methods.
apoptosis in representative AR27-derived hybrids. As shown in Table 3, hybrid cell clones between AR27 and either nontransformed or myc-transformed Rat1A underwent apoptosis in serum-free medium to about the same extent as the parental RMN8 cell line. In order to determine whether the fusion protocol itself, or a dosage-related phenomenon, might account for these observations, we generated hybrids between AR27 itself (see Materials and Methods). By qualitative assessment, AR27 self-hybrids remained as resistant to serum deprivation-induced apoptosis as their unfused counterparts (data not shown). Our results are therefore consistent with the complementation of a recessive-acting mutation in a gene which confers resistance to growth factor deprivation-induced cell death in AR27. Alternatively, a dominant negative mutation might lead to resistance to cell death, and be complemented by a normal gene product in a dosage-dependent fashion (see Discussion). A variety of genotoxic agents, including chemotherapeutic drugs and radiation, have been shown to induce apoptosis through p53-dependent mechanisms [37, 38].
FIG. 5. Western blot analysis of c-myc expression. Nuclear extracts (75 mg) from Rat1A (lane 1), RMN8 (lane 2), and AR27 (lane 3) cells were separated by SDS–PAGE, transferred to nitrocellulose, and assayed for c-Myc using anti-Myc antibody 9E10 and the Amersham ECL detection system.
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Recent studies have implicated wild-type p53 as an essential component in myc-mediated apoptosis [11, 39]. Furthermore, loss of function mutations in p53 have been associated with abrogation of cell death in a number of experimental systems (reviewed in Ref. [40]). We therefore considered the p53 tumor suppressor as a potential candidate for the mutation conferring resistance to apoptosis in AR27. To address this possi-
FIG. 6. Cell viability of Rat1A derivatives generated by proviral rescue. Viability of Rat1A and pooled cell populations derived by proviral rescue was assessed by trypan blue exclusion at various intervals following serum withdrawal. Percentage viability was calculated as described under Materials and Methods. Results are the average from two independent experiments.
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FIG. 7. Induction of apoptosis by staurosporine in parental and apoptosis-resistant cells. Cells were seeded at 5 1 104 per 100-mm dish in complete medium supplemented with 22 nM (L) or 50 nM (H) staurosporine for 48 h. Apoptosis was quantitated by acridine orange staining as described under Materials and Methods. Error bars represent standard deviation from the mean.
bility, we determined the status of the p53 gene product in AR27 as well as in the parental RMN8 cell line. Total cell extracts were prepared from these cells, and p53 protein was immunoprecipitated using antibodies that distinguish between wild-type and mutant conformations. Electrophoretically resolved proteins were transferred to nitrocellulose and immunoblotted with a pan-specific antibody (Pab 421) which recognizes both wild-type and mutant p53. As shown in Fig. 8, the wildtype specific antibody (Pab 246) immunoprecipitated similar amounts of p53 in RMN8 (lane 2) and AR27 (lane 5), whereas the mutant-specific antibody (Pab 240) detected negligible amounts of p53 in both cell lines (lanes 1 and 4). The absence of overaccumulation of p53 in AR27 as compared to its parent, which might be expected based upon the greater stability characteristic of many mutant p53 proteins [41], further supports the interpretation that AR27 expresses only wildtype p53. The pan-specific antibody (Pab 421) detected slightly higher levels of p53 as compared to those obtained with the wild-type specific antibody. However, this was true for both parental (lane 3) and apoptosisresistant cells (lane 6) and is most likely due to the greater affinity of the pan-specific antibody for p53. Therefore, we believe that mutation of the gene for p53 itself is unlikely to account for resistance to apoptosis in AR27. DISCUSSION
It has been widely speculated that apoptosis may serve a protective role in preventing tumorigenicity elicited by deregulated proto-oncogene expression [19]. Specifically, transformed cells, having acquired oncogenic mutations resulting in unscheduled division, are
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eliminated by the activation of a preexisting apoptotic pathway. Although the precise mechanism by which this program is triggered in vivo remains unclear, activation of apoptosis may be a response to adverse growth signals impinging upon the proliferative efforts of neoplastic cells. Such signals might include, but are not limited to, surveillance cytokines such as Fas ligand and tumor necrosis factors [42], as well as angiogenic inhibitors [43] causing inadequate neovascularization and consequent nutrient limitation. In contrast, tumor progression would by necessity require secondary genetic events that abrogate the apoptotic pathway. This notion has been sustained by previous findings demonstrating inhibition of cell death in transformed cells by known dominant-acting modulators of apoptosis, including bcl2, E1B 19K, and mutant p53 (reviewed in Ref. [44]). In the present study, we provide further evidence in support of this phenomenon through the isolation of a rare mutant of c-myc-transformed Rat1A fibroblasts, designated AR27, selected by virtue of its prolonged survival in serum-free medium. The unique characteristics of this cell line are reinforced by the fact that other clones that survived the initial selection failed to exhibit increased viability in a stable fashion. Direct assessment of the cell death phenotype correlates strongly with data obtained from viability assays and confirms that AR27 cells are resistant to growth factor deprivation-induced apoptosis. Most significantly, the mechanism by which resistance to apoptosis arose in this cell line appears to be a novel one, involving a recessive-acting mutation (see below). We have demonstrated unaltered expression of the single human c-myc provirus in AR27 cells, at both the RNA and protein level, consistent with retention of the transformed cell phenotype. Furthermore, we have rescued the proviral transcript by pseudotyping with murine leukemia virus and shown that the c-myc allele retains wild-type ability to mediate transformation and TABLE 3 Apoptosis Assay of AR27 Hybrids % Apoptosisa Cell line Rat1A RMN8 AR27 AR27 AR27 AR27 AR27
1 1 1 1
Rat1A/hyg-1 Rat1A/hyg-2 Rat1A/hyg-3 Rat1A/hyg-4
AR27 1 myc/hyg-1 AR27 1 myc/hyg-2
10% serum
0% serum
3 8 2
4 33 8
8 5 9 5
19 21 18 42
11 6
20 25
a Percentage apoptosis as determined by acridine orange staining after 24 h of culture in complete or serum-free medium.
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apoptosis in Rat1A fibroblasts. On the basis of these findings, we conclude that the apoptosis-resistant phenotype of AR27 is consistent with a mutation in a cellular gene other than c-myc itself. It has been reported that dimerization of c-Myc with its DNA-binding partner Max is required for myc-mediated transformation and apoptosis [17, 18]. It is conceivable that a quantitative alteration in Myc–Max interaction might interfere with the apoptotic function of c-myc without affecting its ability to transform cells. We have not detected any significant difference in steady state levels of max RNA or protein between AR27 and parental RMN8 cells (data not shown). However, our studies do not eliminate the possibility of a qualitative change in Max which might alter the ability of the Myc/Max heterodimer to promote apoptosis. We have shown that the apoptosis-sensitive phenotype was restored in somatic cell hybrids generated between AR27 and either nontransformed or c-myctransformed Rat1A cells. We consider it unlikely that restoration of serum deprivation-induced cell death resulted from an artifact of cell fusion or an increase in gene product dosage (e.g., c-myc), given the observation that AR27 self-hybrids retained the apoptosis-resistant phenotype. Our results are therefore compatible with complementation of a recessive (i.e., loss of function) mutation, leading to abrogation of myc-mediated cell death. It is interesting to speculate that Rat1A cells, which have a pseudodiploid karyotype [Dhanaraj and Small, unpublished results], might be functionally hemizygous at the genetic locus responsible for the apoptosis-resistant phenotype. The reduction in activity or dosage of a mediator of cell death, resulting from mutation of one allele, might also diminish the ability of cells to undergo apoptosis. Alternatively, a dominant negative mutation would be compatible with the recessive behavior of this phenotype in cell hybrids. We have considered the possibility that a dominant-acting gene might be lost in intraspecies hybrids, due to the expected random segregation of chromosomes. However, there is no reason a priori to predict that such a gene would be preferentially deleted in several independently derived hybrid cell lines. Our results from somatic cell hybrid analysis suggest that activation of a dominant-acting suppressor of cell death can be excluded as being responsible for the apoptosis-resistant phenotype of AR27. In this context, we have been unable to detect expression of rat bcl2 in AR27 (or its parental cell line) by Northern or Western blot analysis (data not shown). This finding would tend to rule out the involvement of Bcl2-related proteins such as Bax or Bcl-xS, which are thought to act as negative regulators of Bcl-2 [44]. Furthermore, it is unlikely that an alteration in growth factor signaling (e.g., ectopic growth factor production, constitutive activation of a growth factor receptor or downstream effector) could account for a recessive phenotype. We have exam-
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FIG. 8. Immunoblot analysis of p53. Total cell extracts from RMN8 (lanes 1–3) and AR27 (lanes 4–6) cells were immunoprecipitated with the following anti-p53 monoclonal antibodies: mutantspecific pAb 240 (lanes 1 and 4), wild-type specific pAb 246 (lanes 2 and 5) and pan-specific antibody pAb 421 (lanes 3 and 6). Immunoprecipitates were resolved by SDS–PAGE, transferred to nitrocellulose, and assayed for p53 using PAb 421 and ECL detection.
ined whether AR27-conditioned medium, or cocultivation with AR27 cells in serum-free medium, protects parental RMN8 cells from apoptosis, and have found no evidence for any such effect (data not shown). We have determined that AR27 cells, selected for resistance to growth factor withdrawal-induced cell death, also exhibit resistance to induction of apoptosis by the general protein kinase inhibitor staurosporine. At the highest concentration of staurosporine tested, we do observe a significant amount of cell death, inferring that the apoptotic machinery is at least partially functional. Nevertheless, these results suggest that the apoptosis-resistant phenotype may be due to a mutation in a common component of the cell death pathway. There are a number of possible candidates for such a component, including interleukin-1b converting enzyme (ICE)-like cysteine proteases [45], ornithine decarboxylase [46], cyclin A [47, 48], and the p53 tumor suppressor. The wild-type p53 gene product, a positive effector of cell death in a variety of cell types [40], has recently been implicated in the myc-mediated apoptotic pathway [11, 39]. Furthermore, mutation of p53 is associated with decreased cell death from cytotoxic drugs and radiation [49]. We have examined the status of p53 in AR27 and shown it to be wild-type, on the basis of results obtained using conformation-specific antibodies to distinguish wild-type from mutant species. Within the limitations of this approach, we believe that mutation of p53 itself is unlikely to account for the resistant phenotype. In addition, we have seen no evidence for accumulation of p53 in AR27, a characteristic associated with many mutant p53 proteins [41]. We recognize, however, that wild-type p53 conformation is not necessarily synonymous with normal p53 function. Altered subcellular localization (i.e., cytoplasmic rather than nuclear), as well as loss of transcriptional repression activity [28], might be expected to result in defective p53 function with regard to apoptosis. At a minimum, our studies indicate that the point at
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which cell death is inhibited in AR27 must lie downstream of the pathway by which c-myc affects cell proliferation. Further support for this interpretation comes from a complementary genetic approach [Dhanaraj, Marcus, and Small, manuscript in preparation] in which we have isolated morphological revertants of c-myc-transformed Rat1A cells. In every case examined, selection for loss of the transformed phenotype was accompanied by loss of growth factor deprivationinduced cell death. However, it remains to be established whether c-myc mediates these disparate cellular outcomes through overlapping or independent components. It is intriguing to speculate that c-myc differentially promotes mitogenesis and apoptosis by modulating the expression of distinct target genes. Further characterization of AR27 should help to elucidate the mechanism by which c-myc promotes cell death and the role of this process in tumorigenesis. In addition, this cell line will be useful in studying the relationship between development of resistance to apoptosis and physiological processes associated with cell death, including regulation of intracellular calcium [50] and pH [51]. Finally, it has been shown that a variety of agents currently employed for cancer chemotherapy kill tumor cells by inducing apoptosis [52]. In conjunction with other established mechanisms, abrogation of apoptosis has been implicated in the development of multiple antineoplastic drug resistance [53], a significant concern in cancer therapy. We believe that AR27 cells provide an important model system to gain additional insight into this problem. We thank N. Alwis, M. Smela, and Z. Garcia for their technical assistance, and Drs. H.L. Ozer and R. Wieder for their helpful discussions and review of the manuscript. This work was supported by grants awarded to M.B.S. from the National Cancer Institute (NCI) of the National Institutes of Health (NIH, CA53136) and the Walter and Louise Sutcliffe Foundation. S.N.D. is a recipient of a fellowship from the New Jersey Commission on Cancer Research.
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10. Bissonnette, R. P., Echeverri, F., Mahboubi, A., and Green, D. R. (1992) Nature 359, 552–554. 11. Hermeking, H., and Eick, D. (1994) Science 265, 2091–2093. 12. Shi, Y., Glynn, J. M., Guilbert, L. J., Cotter, T. G., Bissonnette, R. P., and Green, D. R. (1992) Science 257, 212–214. 13. Milner, A. E., Grand, R. J. A., Waters, C. M., and Gregory, C. D. (1993) Oncogene 8, 3385–3391. 14. Amati, B., Dalton, S., Brooks, M. W., Littlewood, T. D., Evan, G. I., and Land, H. (1992) Nature 359, 423–426. 15. Kretzner, L., Blackwood, E. M., and Eisenmann, R. N. (1992) Nature 359, 426–429. 16. Amin, C., Wagner, A. J., and Hay, N. (1993) Mol. Cell. Biol. 13, 383–390. 17. Amati, B., Littlewood, T. D., Evan, G. I., and Land, H. (1993) EMBO J. 12, 5083–5087. 18. Bissonnette, R. P., McGahon, A., Mahboubi, A., and Green, D. R. (1994) J. Exp. Med. 180, 2413–2418. 19. Williams, G. T. (1991) Cell 65, 1097–1098. 20. Fanidi, A., Harrington, E. A., and Evan, G. I. (1992) Nature 359, 554–556. 21. Wagner, A. J., Small, M. B., and Hay, N. (1993) Mol. Cell. Biol. 13, 2432–2440. 22. Korah, R. M., Wagner, A. J., Dhanaraj, S. N., Hay, N., and Small, M. B., submitted for publication. 23. Strasser, A., Harris, A. W., Bath, M. L., and Cory, S. (1990) Nature 348, 331–333. 24. Marin, M. C., Hsu, B., Stephens, L. C., Brisbay, S., and McDonnell, T. J. (1995) Exp. Cell Res. 217, 240–247. 25. Harrington, E. A., Bennett, M. R., Fanidi, A., and Evan, G. I. (1994) EMBO J. 13, 3286–3295. 26. Mann, R., Mulligan, R., and Baltimore, D. (1983) Cell 33, 153– 159. 27. Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, M. (1990) Mut. Res. 245, 245–249. 28. Caelles, C., Helmberg, A., and Karin, M. (1994) Nature 370, 220–223. 29. Kizaki, H., Tadakuma, T., Odaka, C., Muramatsu, J., and Ishimura, Y. (1989) J. Immunol. 143, 1790–1794. 30. Huebner, R. J., Hartley, J. W., Rowe, W. P., Lane, W. T., and Capps, W. I. (1966) Proc. Natl. Acad. Sci. USA 56, 1164–1169. 31. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475–1489. 32. Harlow, E., and Lane, D. (1988) in Antibodies: A Laboratory Manual, pp. 464–466, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 33. Davidson, R. L., and Gerald, P. S. (1976) Somatic Cell Genet. 2, 165–176. 34. Mulligan, R. C., and Berg, P. (1980) Science 209, 1422–1427. 35. Eilers, M., Schirm, S., and Bishop, J. M. (1991) EMBO J. 10, 133–141. 36. Bertrand, R., Solary, E., O’Connor, P., Kohn, K. W., and Pommier, Y. (1994) Exp. Cell Res. 211, 314–321. 37. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L., and Wyllie, A. H. (1993) Nature 362, 849– 852. 38. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. (1993) Nature 362, 847–849. 39. Wagner, A. J., Kokontis, J. M., and Hay, N. (1994) Genes Dev. 8, 2817–2830. 40. Oren, M. (1994) Semin. Cancer Biol. 5, 221–227. 41. Levine, A. J., Momand, J., and Finlay, C. A. (1991) Nature 351, 453–456.
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42. Cleveland, J. L., and Ihle, J. N. (1995) Cell 81, 479–482. 43. Holmgren, L., O’Reilly, M. S., and Folkman, J. (1995) Nature Med. 1, 149–153. 44. Oltvai, Z. N., and Korsmeyer, S. J. (1994) Cell 79, 189–192. 45. Vaux, D. L., Haecker, G., and Strasser, A. (1994) Cell 76, 777– 779. 46. Packham, G., and Cleveland, J. L. (1994) Mol. Cell. Biol. 14, 5741–5747. 47. Hoang, A. T., Cohen, K. J., Barrett, J. F., Bergstrom, D. A., and Dang, C. V. (1994) Proc. Natl. Acad. Sci. USA 91, 6875–6879.
48. Meikrantz, W., Gisselbrecht, S., Tam, S. W., and Schlegel, R. (1994) Proc. Natl. Acad. Sci. USA 91, 3754–3758. 49. Stewart, B. W. (1994) J. Natl. Cancer Inst. 86, 1286–1296. 50. Trump, B. F., and Berezesky, I. K. (1995) FASEB J. 9, 219– 228. 51. Barry, M. A., and Eastman, A. (1992) Biochem. Biophys. Res. Commun. 186, 782–789. 52. Hickman, J. A. (1992) Cancer Metastasis Rev. 11, 121–139. 53. Fisher, D. E. (1994) Cell 78, 539–542.
Received October 16, 1995 Revised version received January 19, 1996
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0110
Characterization of c-myc-Transformed Rat Fibroblasts Resistant to Apoptosis Induced by Growth Factor Deprivation SRIDEVI N. DHANARAJ, ALEXANDER M. MARCUS, REJU M. KORAH, KOICHI IWATA,
AND
MICHAEL B. SMALL1
Department of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical School, Newark, New Jersey, 07103-2714
apoptosis (reviewed in Refs. [6, 7]). This property of c-myc has been demonstrated in diverse cell types, including murine myeloid cells [8], primary and established rodent fibroblasts [9 – 11], T-cell hybridomas [12], and Burkitt lymphoma cells [13]. The precise mechanism by which c-myc contributes to the regulation of proliferation and apoptosis remains to be established. Myc, a nuclear-localized DNA-binding phosphoprotein, has been shown to function as a transcriptional activator in vitro [14–16], although its in vivo target genes remain largely unknown. Mutational analysis has revealed that the functional domains of c-Myc required for transformation are also necessary for its ability to mediate apoptosis [9]. Furthermore, dimerization of c-Myc with its DNA-binding partner Max is required for myc-mediated transformation and apoptosis [17, 18]. These results suggest that the transcriptional regulatory activities of c-myc are required for its apoptotic functions. It has been proposed that apoptosis may serve a protective role in preventing tumorigenicity elicited by deregulated Myc expression [19]. Tumor progression would therefore require a mechanism for abrogation of the myc-mediated cell death pathway. A number of possible secondary genetic events could account for such an outcome. First, an alteration in c-Myc (or Max) might impair the apoptotic function of c-Myc without affecting its mitogenic properties. Second, activation of a gene encoding an anti-apoptotic protein might be involved. In this regard, it has been shown that overexpression of human Bcl-2 [10, 20, 21], Bcl-xL [22], or the adenovirus E1B 19K protein [22] is capable of blocking myc-mediated apoptosis without affecting the transformed phenotype. In addition, Myc and Bcl-2 appear to act synergistically in several tumor cell systems [23, 24]. Third, it has been demonstrated that serum cytokines, in particular the insulin-like growth factors, act as survival signals to protect cells against myc-mediated apoptosis [25]. A mutation leading to constitutive activation of the signal transduction pathways through which these factors operate could therefore lead to resistance to apoptosis induced by growth factor deprivation. Finally, apoptosis could be blocked by inactivation of an integral component in the cell death machinery. In this study, we have sought to further examine
Under appropriate conditions (e.g., growth factor withdrawal), the deregulated expression of c-myc in rodent fibroblasts leads to substantial cell death due to apoptosis. To better understand this process, we selected for c-myc-transformed Rat1A fibroblasts that were resistant to growth factor deprivation-induced cell death. One clonal isolate exhibited prolonged survival in serum-free medium and displayed reduced levels of apoptosis-related DNA fragmentation. These cells were also resistant to induction of apoptosis by the protein kinase inhibitor staurosporine. They retained a transformed cell phenotype and expressed the proviral human c-myc allele in an unaltered fashion, strongly indicating that the mutation of a cellular gene other than c-myc accounts for the apoptosis-resistant phenotype. The results of somatic cell hybrid analysis of this cell line are consistent with a recessive mutation. Our findings suggest a novel mechanism for abrogation of apoptosis in neoplastic cells and provide a model system for the study of its role in tumorigenesis and resistance to antineoplastic therapy. q 1996 Academic Press, Inc.
INTRODUCTION
The c-myc proto-oncogene has long been recognized as an important regulator of normal and malignant cell growth (reviewed in Refs. [1 – 3]). Expression of cmyc serves as an essential signal for entry and progression through the cell cycle and as a modulator of cell differentiation. Deregulation of c-myc is strongly implicated in the pathogenesis of a variety of human tumors, and its in vitro transforming activity has been clearly demonstrated [4, 5]. Most recently, it has become clear that the c-Myc oncoprotein has pleiotropic functions that act in opposition to its role as a mediator of cell proliferation. Specifically, it has been shown that, under appropriate conditions (e.g., growth factor deprivation), the deregulated expression of c-myc can promote cell death by a process attributable to 1
To whom correspondence and reprint requests should be addressed. Fax: (201) 982-3644. 52
0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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the mechanisms involved in myc-mediated apoptosis through the selection of apoptosis-resistant mutants of c-myc-transformed Rat1A fibroblasts. A clonal isolate exhibited resistance to cell death induced by both serum withdrawal and the general protein kinase inhibitor staurosporine, while retaining the transformed cell phenotype. On the basis of several criteria, we believe that a recessive-acting mutation in a cellular gene other than c-myc itself is responsible for the apoptosisresistant phenotype. Our findings suggest a novel mechanism for abrogation of apoptosis in neoplastic cells and provide a model system for the study of its role in tumorigenesis and resistance to antineoplastic therapy. MATERIALS AND METHODS Cell lines and cell culture. The parental transformed cell line used in these studies, designated RMN8, was generated by infection of Rat1A cells with a retroviral stock derived by transfection of C2 packaging cells [26] with the retroviral vector pMV6myc [21]. pMV6myc expresses a human c-myc cDNA from a murine leukemia viral long terminal repeat (LTR) and the bacterial gene that confers resistance to the antibiotic G418 under the control of an internal thymidine kinase promoter. RMN8 cells were initially selected in complete medium (see below) containing 800 mg/ml G418 and then recloned in 0.35% agarose (SeaPlaque). The myc/bcl2 polyclonal cell line was described previously [21]. For cell fusion studies, hygromycin B-resistant derivatives of nontransformed (Rat1A/hyg) and cmyc-transformed (myc/hyg) Rat1A cells were generated by infection with pMV12 and pMV12myc retroviruses, respectively [21]. Cell lines were maintained in Dulbecco’s modified Eagle’s medium/ F-10 (1:1) containing 10% newborn calf serum (complete medium) at 377C in 7.5% CO2 . Anchorage-independent growth was measured as described [4]. Two hundred cells were resuspended in agarose (containing complete medium) and overlaid into triplicate 60-mm dishes that were visually inspected for colony formation after incubation at 377C for 14 to 21 days. Isolation of apoptosis-resistant cells. RMN8 cells were initially seeded in fifty 100-mm dishes at 105 cells per dish in complete medium. The following day, cells were washed with phosphate-buffered saline (PBS), refed with serum-free medium, and incubated at 377C for 6 days. These conditions have previously been determined to result in virtually complete death of these cells by apoptosis. Plates were then refed with complete medium to allow viable cells to recover and form colonies. One week later, 35 surviving colonies were isolated and expanded into mass culture in medium containing 400 mg/ ml G418, indicative of continued expression of the integrated c-myc provirus. All 35 colonies were then reexamined for enhanced viability in serum-free media, as assessed qualitatively by increase in cell number or decrease in floating (detached) cells. Determination of cell viability and apoptosis. For viability and apoptosis assays, cells were seeded in complete medium at 5 1 104 cells per 100-mm dish. Twenty-four hours later, duplicate dishes were washed with PBS, refed with complete or serum-free medium, with and without 22 or 50 nM staurosporine (Calbiochem), and cultured for a period of 72 h. Detached and adherent cells (harvested by trypsinization) were resuspended in PBS and counted in a Royco cell counter (total cell counts). Total viable cell number was determined by trypan blue exclusion at various times after induction of cell death, as described [21]. For quantitation of apoptosis, detached and adherent cells were pooled, washed in PBS, and collected by centrifugation at 1200g for 10 min. Cells were resuspended in 1:10 volume of cold fixing solution (PBS with 5% NCS and 1% formaldehyde). Ten microliters of fixed
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cell suspension was placed on an acridine orange-coated slide, sealed with a coverslip, and observed by fluorescent microscopy [27]. The average number of apoptotic cells (i.e., those displaying chromatin condensation or formation of apoptotic bodies) was determined by counting 200 cells (in each of three separate fields) on triplicate slides. The percentage of apoptotic cells was calculated by dividing the number of apoptotic cells by total cell number and multiplying by 100. For DNA fragmentation analysis, total DNA was isolated from subconfluent cell populations by lysis in buffer containing 0.5% SDS, 10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 50 mM EDTA, and 500 mg/ml proteinase K followed by phenol–chloroform extraction and ethanol precipitation. One hundred nanograms of each DNA sample was end-labeled with [a-32P]dideoxyATP using terminal transferase [28]. Following termination of the labeling reaction, DNA was ethanol precipitated, collected by centrifugation, and resuspended in 20 ml of 10 mM Tris–HCl, 1 mM EDTA, pH 7.5. Thirty-five nanograms of each labeled DNA was electrophoresed on a 1.8% agarose gel in Tris–acetate–EDTA buffer at 50 V for 6 h. The gel was fixed in 10% trichloroacetic acid for 30 min, rinsed in distilled water, vacuumdried, and exposed to autoradiographic film overnight. Fluorimetric quantitation of low-molecular-weight DNA, isolated by detergent lysis and differential centrifugation, was performed by an adaptation of the method as described in Ref. [29]. Flow cytometric analysis. Cells were harvested by trypsinization and washed three times in ice-cold PBS (Mg2/-Ca2/-free). Cells in 0.9 ml of PBS were passed through a 25-gauge needle and fixed by dropwise addition into 2.1 ml of ice-cold 100% ethanol. Approximately 106 fixed cells were resuspended in 0.9 ml PBS containing 7.5 mg/ml DNase-free RNase A. Cells were incubated at 377C for 1 h and stained with 50 mg/ml propidium iodide. Cell cycle distributions were determined by analysis of 104 cells using the CellFIT program on a FACScan flow cytometer (Becton-Dickinson). Proviral rescue by superinfection with murine leukemia virus. Cells were assayed for the presence of a functional human c-myc gene by superinfection with Moloney murine leukemia virus (MLV) and generation of pseudotype retrovirus [30]. Cells were seeded at 5 1 105 per 100-mm dish, infected the following day with 2 ml of MLV-containing medium (a gift of M. Roth, UMDNJ-Robert Wood Johnson Medical School) supplemented with 8 mg/ml Polybrene, and then refed 4 h later with complete medium. Medium was collected from superinfected cells 48 h postinfection and passed through a 0.45-mm Millipore filter, and 2 ml of this medium was used to infect Rat1A cells, seeded at 5 1 105 per 100-mm dish, as described above. Forty-eight hours postinfection, infected Rat1A cells were trypsinized and reseeded into complete medium containing 400 mg/ml G418. After 7 to 10 days, dishes were examined for the presence of G418resistant colonies. Approximately 100 colonies from each of two separate dishes were pooled to generate independent cell populations (e.g., AR27-RP1 and AR27-RP2), and viability and anchorage dependence assays were performed on each pool as described previously. Preparation of nuclear and whole cell extracts. Nuclear extracts were prepared as described [31]. Protein concentrations were determined by the Bradford assay and extracts were stored at 0707C. Whole cell extracts (for p53 analyses) were prepared by lysis of one 100-mm subconfluent dish in 1 ml of lysis buffer (25 mM Tris– HCl, pH 7.5, 100 mM NaCl, 2% NP-40, 0.2% SDS, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mg/ ml aprotinin, and 50 mM leupeptin). Lysates were incubated for 30 min at 47C and then centrifuged (12,000g) for 15 min at 47C. The supernatant was collected and used for p53 immunoprecipitation. Analysis of c-Myc and p53 protein. One hundred micrograms of protein from nuclear extracts in sample buffer containing 33% glycerol, 0.3 M DTT, 6.7% SDS, and 0.01% bromophenol blue was electrophoresed on a 10% SDS–polyacrylamide gel at 30 V for 18 h. Separated proteins were transferred to 0.2-mm nitrocellulose (Schleicher & Schuell) using a Hoeffer transfer apparatus. Nitrocellulose filters were blocked for 1 h with 5% (w/v) nonfat dry milk in
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buffer containing 10 mM Tris–HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20 (TBST), and then incubated with mouse monoclonal antibody 9E10 (Oncogene Science), diluted 1:20, for detection of c-Myc protein. Filters were washed in TBST and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat antimouse antibody. Filters were developed in ECL detection reagent (Amersham) for 1 min and exposed to X-ray film for 10 s. For determination of p53 status, whole cell extracts (300 ml) were immunoprecipitated overnight at 47C with 1 mg of each of the following anti-p53 monoclonal antibodies (Oncogene Science): wild-type specific Pab 246, mutant-specific Pab 240, or pan-specific Pab 421 (which detects both wild-type and mutant p53). Protein G-Plus agarose beads (Oncogene Science) were added and incubated for 30 min at 47C as described [32]. The immunoprecipitates were washed extensively in lysis buffer, boiled in SDS sample buffer, and then resolved by SDS–polyacrylamide gel electrophoresis and immunoblotting as described above. p53 was detected using Pab 421 as the primary antibody and an anti-mouse k light chain peroxidase-conjugated secondary antibody (Zymed), followed by ECL detection. Somatic cell hybrid analysis. AR27 (2.5 1 105) cells were fused to Rat1A/hyg or myc/hyg (2.5 1 106) cells by treatment with 50% w/v polyethylene glycol (PEG) 1500 for 1 min [33]. Cells were then reseeded into complete medium containing G418 (400 mg/ml) and hygromycin B (200 mg/ml). For production of self-hybrids, AR27 derivatives resistant to hygromycin B or expressing the bacterial gene gpt were generated by calcium phosphate-mediated transfection with the plasmids pSV2gpt [34] or puc/hyg [4], respectively. Following PEG fusion, AR27 self-hybrids were selected in complete medium containing hygromycin B (200 mg/ml), 25 mg/ml mycophenolic acid, and 75 mg/ml xanthine. Hybrid colonies were isolated, expanded into mass culture, and assessed for serum deprivation-induced apoptosis by acridine orange staining as described above.
RESULTS
Isolation of Apoptosis-Resistant Cell Line AR27 Derivatives of the established rodent fibroblast cell line Rat1A transformed by human c-myc undergo apoptosis when deprived of serum growth factors in vitro [9, 21]. To further understand the mechanisms involved in myc-mediated apoptosis, we undertook to isolate apoptosis-resistant mutants of myc-transformed Rat1A by selection of cells capable of prolonged survival in serum-free medium. This strategy would not be expected to have a bias for or against retention of the transformed phenotype. The parental cell line used in these studies, designated RMN8, expresses a human c-myc allele introduced by infection of Rat1A cells with the retrovirus pMV6myc, which also encodes the bacterial gene conferring resistance to the antibiotic G418 (see Materials and Methods). RMN8 cells, selected initially for G418 resistance, display a transformed cell morphology and are able to form colonies in soft agarose. Southern analysis (data not shown) demonstrated that these cells harbor a single pMV6myc provirus. A total of 5 1 106 RMN8 cells, initially seeded at 105 cells per 100-mm dish in complete medium, were washed with PBS, refed with serum-free medium, and incubated at 377C for 6 days. Under these conditions, the vast majority of cells underwent programmed cell death and detached from the dish, a phenotype associ-
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ated with apoptosis in these cells [9]. Plates were then refed with complete medium to allow surviving cells to recover and form colonies. One week later, 35 colonies were isolated and expanded into mass culture in medium containing the antibiotic G418, indicative of continued expression of the integrated c-myc provirus. All 35 colonies were then reexamined individually for viability in serum-free medium. One clone, designated AR27, displayed prolonged survival in serum-free medium relative to the parental RMN8 cell line, as assessed qualitatively by either an increase in cell number or a decrease in detached cells. Unlike the other clones tested, this phenotype was stable upon continued passage in culture and was independent of cell density (compare Figs. 1A and B). Furthermore, AR27 cells exhibit a transformed cell morphology in complete medium indistinguishable from that of parental RMN8 cells, although they appear somewhat flatter and less refractile in serum-free medium (see Fig. 1A). Maintenance of the transformed cell phenotype of AR27 was also confirmed by assay for growth in soft agarose. As shown in Table 1, AR27 cells form anchorage-independent colonies (as well as colonies in monolayer culture) with efficiencies comparable to the RMN8 cells. Cell cycle analysis by flow cytometry (see Table 2) confirmed that AR27 displays a cycling cell distribution after 24 h in serum-free medium, indistinguishable from parental RMN8 cells. We determined total viable cell counts (using trypan blue exclusion) of AR27 in complete and serum-free media, in comparison to the parental RMN8 cell line and another clone (AR35) which had emerged from the initial selection. Figure 2A shows that the growth rates in complete medium for AR27 and AR35 are similar to that of RMN8. In serum-free medium (Fig. 2B), AR27 cells continued to proliferate, albeit at a slower rate than in complete medium, and retained nearly 100% viability following 72 h of serum withdrawal. In contrast, parental RMN8 and AR35 cells exhibited only a slight increase in cell number over the same time period. These results reflect the documented ability of deregulated c-myc expression to promote cell proliferation in the absence of serum growth factors [35], offset in the case of RMN8 and AR35 cells by a 70% decrease in cell viability. Thus, the greater net increase in cell number of AR27 in serum-free medium, relative to that of RMN8 and AR35, is attributable to enhanced viability, rather than increased proliferative ability. Resistance of AR27 to Serum Deprivation-Induced Apoptosis We next sought to determine whether the increased viability in serum-free medium displayed by AR27 was due to resistance to apoptotic cell death induced by growth factor withdrawal. The degradation of nuclear DNA into fragments of nucleosome length units (or
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FIG. 1. Cell morphology in complete and serum-free medium. Parental RMN8 and apoptosis-resistant AR27 cells were seeded at 1 1 105 (A) or 1 1 104 (B) per 100-mm dish. Cells were photographed after 5 days of growth factor deprivation. (Original magnification, 1100.)
multiples thereof) is widely regarded as a reliable biomarker for apoptosis (reviewed in Ref. [6]). We therefore analyzed AR27 cells for evidence of apoptosis-specific DNA fragmentation (i.e., nucleosome ladders) upon culture in serum-free medium. For comparative purposes, we included in our analysis nontransformed Rat1A and parental RMN8 cells, as well as a myctransformed Rat1A derivative that overexpresses the human bcl2 gene (myc/bcl2) and is resistant to apoptosis [21]. Total DNA was isolated from both adherent and detached cells grown in either complete or serum-free medium for 48 h. Equal amounts of DNA from each cell line under both growth conditions, radiolabeled by terminal transferase, were resolved by agar-
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ose gel electrophoresis. As shown in Fig. 3, AR27 exhibits significantly reduced levels of nucleosome-unitlength fragments in serum-free medium (lane 6), as compared to parental RMN8 cells grown under the same conditions (lane 4). The level of DNA fragmentation in AR27 appears to be even lower than that observed in myc/bcl2 cells (lane 8). Comparable results were obtained by fluorimetric quantitation of Hoechst 33258-stained low-molecular-weight DNA isolated by differential centrifugation of detergent-lysed cells (data not shown). We also examined the same cell lines at various times after serum deprivation for morphological changes characteristic of apoptosis using fluorescence
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TABLE 1 Efficiency of Colony Formationa Cell line
Agarose
Monolayer
Rat1A RMN8 AR27 RMN8-RP1 RMN8-RP2 AR27-RP1 AR27-RP2
õ1 26 22 28 25 19 21
40 28 26 38 35 26 31
a Efficiency of colony formation as percentage of input cells; 200 cells were seeded in agarose or monolayer in duplicate dishes as described under Materials and Methods.
microscopy of acridine orange-stained cells. This method of analysis allowed us to more precisely quantify the percentage of apoptotic cells at specific time points. As shown in Fig. 4, AR27 cells are as resistant as myc/bcl2 cells, even 72 h after withdrawal of growth factors. In contrast, greater than 70% of the parental RMN8 cells exhibit apoptotic changes by the same time point. Thus, all three methods used to measure apoptosis, in conjunction with cell proliferation and viability assays, have yielded consistent data that AR27 cells are resistant to serum deprivation-induced cell death. Retention of Functional c-myc Expression in AR27 As described above, AR27 cells are resistant to apoptosis induced by growth factor withdrawal, yet continue to manifest a transformed cell phenotype characteristic of the RMN8 cell line from which they were derived. The persistence of the transformed phenotype would suggest that these cells continue to express functional c-myc RNA and protein. Southern analysis (data not shown) revealed no apparent alteration in the structure of the integrated pMV6myc proviral DNA. However, it has previously been shown that transformation and apoptosis mediated by c-myc are dosage-dependent phenomena [4, 9]. It was conceivable that reduced c-myc expression in AR27 might account for resistance to myc-mediated apoptosis, without affecting the transformed phenotype. To address this possibility, we compared the levels of human c-Myc protein in nuclear extracts from parental RMN8 and AR27 cells by Western blot analysis. As shown in Fig. 5, AR27 (lane 3) expresses steady state levels of c-Myc protein equivalent to those of RMN8 (lane 2). Northern blot and RNase protection analysis of AR27 also revealed no alteration in c-myc expression at the transcriptional level (data not shown). Similar results were found in cells cultured in complete medium as well as in serumfree medium, consistent with the absence of a serum responsive element in the retroviral promoter driving c-myc expression.
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To assess the functional status of the human c-myc allele in AR27, we superinfected these cells, in parallel with the parental RMN8 cell line, with Moloney murine leukemia virus (MLV), giving rise to cells producing a mixed population of wild-type MLV and pseudotype retrovirus [30] containing the human c-myc proviral transcript. Tissue culture supernatants from the MLVinfected cell lines were used to infect nontransformed Rat1A cells, which were then selected by colony formation in medium containing the antibiotic G418. G418resistant colonies were pooled, expanded into mass culture, and examined for phenotypic transformation as well as for their ability to undergo apoptosis upon serum withdrawal. Pooled cell populations (AR27-RP1 and RP2) derived by infection with rescued virus from AR27 displayed a transformed cell morphology and an anchorage-independent phenotype (see Table 1) no different from cells generated by infection with rescued virus from RMN8 (RMN8-RP1 and RP2). As shown in Fig. 6, all pooled rescue cell lines exhibited a loss of cell viability upon serum withdrawal, as measured by trypan blue exclusion, indistinguishable from one another in both magnitude as well as time course. Microscopic examination of these cells (data not shown) confirmed that cell death was due to apoptosis. On the basis of these findings, we conclude that the human c-myc allele resident in AR27 is unaltered in expression and function. Thus, the apoptosis-resistant phenotype is consistent with a mutation in a cellular gene other than c-myc itself. These results also provide evidence for independent components in the pathways by which c-myc mediates transformation and apoptosis (see Discussion). Cross-Resistance of AR27 to Induction of Apoptosis by Staurosporine We considered the possibility that AR27, selected for resistance to serum deprivation-induced apoptosis, might also be resistant to induction of programmed cell death by other agents. Cross resistance to multiple
TABLE 2 Cell Cycle Analysis in Complete and Serum-Free Medium % of cells (mean { SD) in: Cell line
Serum
RAT1A RMN8 AR27 RAT1A RMN8 AR27
/ / / 0 0 0
G0/G1 34.8 38.6 39.9 78.2 55.9 57.8
{ { { { { {
0.1 1.7 0.1 0.4 0.1 0.0
S 51.4 43.6 44.5 15.0 33.3 30.6
{ { { { { {
G2/M 0.0 3.7 0.1 0.0 0.0 0.1
13.8 17.8 15.8 6.8 10.8 11.7
{ { { { { {
0.1 2.1 0.1 0.4 0.0 0.1
Note. SD, standard deviation; data represent two independent experiments.
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FIG. 2. Growth curves of parental and apoptosis-resistant cells. Cells seeded at 5 1 104 per 100-mm dish were grown in complete (A) or serum-free (B) medium. Total viable cell counts were determined as described under Materials and Methods. Error bars represent standard deviation from the mean.
inducers of apoptosis might suggest that the mutation responsible for apoptosis resistance directly involves a component of the cell death machinery. We chose to examine the response of AR27 to treatment with the general protein kinase inhibitor staurosporine, which has been shown to induce apoptosis in a wide variety of cell types [36]. AR27 cells, along with RMN8 and myc/bcl2 cells, were cultured in complete medium in
the absence and presence of 22 nM (low) and 50 nM (high) staurosporine. Apoptosis, as quantitated by acridine orange staining, was measured after 48 h of treatment under these conditions. As shown in Fig. 7, parental RMN8 cells display significant cell death at both concentrations tested. In contrast, AR27 cells are markedly resistant to staurosporine-induced cell death at 22 nM, although they display only partial resistance at the higher concentration. In comparison, myc/bcl2 cells are protected at both low and high staurosporine levels. These results suggest that the apoptosis-resistant phenotype of AR27 may involve a mutation in a component of the cell death pathway common to induction by both growth factor withdrawal and staurosporine. Measurements of cell viability, as determined by trypan blue exclusion, parallel the results we have obtained by acridine orange staining (data not shown). Genetic Analysis of the Apoptosis-Resistant Phenotype of AR27
FIG. 3. Analysis of DNA fragmentation. Total DNA was isolated from adherent and detached cells, radiolabeled with terminal transferase, and resolved by electrophoresis on 1.8% agarose gel. Rat1A (lanes 1 and 2), RMN8 (lanes 3 and 4), AR27 (lanes 5 and 6), and Myc/Bcl2 (lanes 7 and 8) cells were grown for 48 h in complete (lanes 1, 3, 5, and 7) or serum-free (lanes 2, 4, 6, and 8) medium.
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In order to determine the genetic nature (i.e., dominance or recessiveness) of the apoptosis-resistant phenotype of AR27, we generated somatic cell hybrids between AR27, which is resistant to the antibiotic G418, and both nontransformed and c-myc-transformed Rat1A cell lines expressing the bacterial gene encoding resistance to the antibiotic hygromycin B (see Materials and Methods). Hybrid cells were selected by colony formation in complete medium supplemented with G418 and hygromycin B, conditions shown to kill the unhybridized parental cells. Individual colonies were tested for survival in serum-free medium. Qualitative observations suggested that the apoptosis-sensitive phenotype was restored in the majority of hybrids examined. We then used fluorescence microscopy of acridine orange-stained cells to quantitate the degree of
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FIG. 4. Quantitation of apoptosis in parental and apoptosis-resistant cells. Adherent and detached cells were stained with acridine orange following culture in complete or serum-free medium for 0, 12, 24, 48, and 72 h and observed by fluorescent microscopy. Percentage apoptosis was calculated as described under Materials and Methods.
apoptosis in representative AR27-derived hybrids. As shown in Table 3, hybrid cell clones between AR27 and either nontransformed or myc-transformed Rat1A underwent apoptosis in serum-free medium to about the same extent as the parental RMN8 cell line. In order to determine whether the fusion protocol itself, or a dosage-related phenomenon, might account for these observations, we generated hybrids between AR27 itself (see Materials and Methods). By qualitative assessment, AR27 self-hybrids remained as resistant to serum deprivation-induced apoptosis as their unfused counterparts (data not shown). Our results are therefore consistent with the complementation of a recessive-acting mutation in a gene which confers resistance to growth factor deprivation-induced cell death in AR27. Alternatively, a dominant negative mutation might lead to resistance to cell death, and be complemented by a normal gene product in a dosage-dependent fashion (see Discussion). A variety of genotoxic agents, including chemotherapeutic drugs and radiation, have been shown to induce apoptosis through p53-dependent mechanisms [37, 38].
FIG. 5. Western blot analysis of c-myc expression. Nuclear extracts (75 mg) from Rat1A (lane 1), RMN8 (lane 2), and AR27 (lane 3) cells were separated by SDS–PAGE, transferred to nitrocellulose, and assayed for c-Myc using anti-Myc antibody 9E10 and the Amersham ECL detection system.
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Recent studies have implicated wild-type p53 as an essential component in myc-mediated apoptosis [11, 39]. Furthermore, loss of function mutations in p53 have been associated with abrogation of cell death in a number of experimental systems (reviewed in Ref. [40]). We therefore considered the p53 tumor suppressor as a potential candidate for the mutation conferring resistance to apoptosis in AR27. To address this possi-
FIG. 6. Cell viability of Rat1A derivatives generated by proviral rescue. Viability of Rat1A and pooled cell populations derived by proviral rescue was assessed by trypan blue exclusion at various intervals following serum withdrawal. Percentage viability was calculated as described under Materials and Methods. Results are the average from two independent experiments.
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FIG. 7. Induction of apoptosis by staurosporine in parental and apoptosis-resistant cells. Cells were seeded at 5 1 104 per 100-mm dish in complete medium supplemented with 22 nM (L) or 50 nM (H) staurosporine for 48 h. Apoptosis was quantitated by acridine orange staining as described under Materials and Methods. Error bars represent standard deviation from the mean.
bility, we determined the status of the p53 gene product in AR27 as well as in the parental RMN8 cell line. Total cell extracts were prepared from these cells, and p53 protein was immunoprecipitated using antibodies that distinguish between wild-type and mutant conformations. Electrophoretically resolved proteins were transferred to nitrocellulose and immunoblotted with a pan-specific antibody (Pab 421) which recognizes both wild-type and mutant p53. As shown in Fig. 8, the wildtype specific antibody (Pab 246) immunoprecipitated similar amounts of p53 in RMN8 (lane 2) and AR27 (lane 5), whereas the mutant-specific antibody (Pab 240) detected negligible amounts of p53 in both cell lines (lanes 1 and 4). The absence of overaccumulation of p53 in AR27 as compared to its parent, which might be expected based upon the greater stability characteristic of many mutant p53 proteins [41], further supports the interpretation that AR27 expresses only wildtype p53. The pan-specific antibody (Pab 421) detected slightly higher levels of p53 as compared to those obtained with the wild-type specific antibody. However, this was true for both parental (lane 3) and apoptosisresistant cells (lane 6) and is most likely due to the greater affinity of the pan-specific antibody for p53. Therefore, we believe that mutation of the gene for p53 itself is unlikely to account for resistance to apoptosis in AR27. DISCUSSION
It has been widely speculated that apoptosis may serve a protective role in preventing tumorigenicity elicited by deregulated proto-oncogene expression [19]. Specifically, transformed cells, having acquired oncogenic mutations resulting in unscheduled division, are
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eliminated by the activation of a preexisting apoptotic pathway. Although the precise mechanism by which this program is triggered in vivo remains unclear, activation of apoptosis may be a response to adverse growth signals impinging upon the proliferative efforts of neoplastic cells. Such signals might include, but are not limited to, surveillance cytokines such as Fas ligand and tumor necrosis factors [42], as well as angiogenic inhibitors [43] causing inadequate neovascularization and consequent nutrient limitation. In contrast, tumor progression would by necessity require secondary genetic events that abrogate the apoptotic pathway. This notion has been sustained by previous findings demonstrating inhibition of cell death in transformed cells by known dominant-acting modulators of apoptosis, including bcl2, E1B 19K, and mutant p53 (reviewed in Ref. [44]). In the present study, we provide further evidence in support of this phenomenon through the isolation of a rare mutant of c-myc-transformed Rat1A fibroblasts, designated AR27, selected by virtue of its prolonged survival in serum-free medium. The unique characteristics of this cell line are reinforced by the fact that other clones that survived the initial selection failed to exhibit increased viability in a stable fashion. Direct assessment of the cell death phenotype correlates strongly with data obtained from viability assays and confirms that AR27 cells are resistant to growth factor deprivation-induced apoptosis. Most significantly, the mechanism by which resistance to apoptosis arose in this cell line appears to be a novel one, involving a recessive-acting mutation (see below). We have demonstrated unaltered expression of the single human c-myc provirus in AR27 cells, at both the RNA and protein level, consistent with retention of the transformed cell phenotype. Furthermore, we have rescued the proviral transcript by pseudotyping with murine leukemia virus and shown that the c-myc allele retains wild-type ability to mediate transformation and TABLE 3 Apoptosis Assay of AR27 Hybrids % Apoptosisa Cell line Rat1A RMN8 AR27 AR27 AR27 AR27 AR27
1 1 1 1
Rat1A/hyg-1 Rat1A/hyg-2 Rat1A/hyg-3 Rat1A/hyg-4
AR27 1 myc/hyg-1 AR27 1 myc/hyg-2
10% serum
0% serum
3 8 2
4 33 8
8 5 9 5
19 21 18 42
11 6
20 25
a Percentage apoptosis as determined by acridine orange staining after 24 h of culture in complete or serum-free medium.
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apoptosis in Rat1A fibroblasts. On the basis of these findings, we conclude that the apoptosis-resistant phenotype of AR27 is consistent with a mutation in a cellular gene other than c-myc itself. It has been reported that dimerization of c-Myc with its DNA-binding partner Max is required for myc-mediated transformation and apoptosis [17, 18]. It is conceivable that a quantitative alteration in Myc–Max interaction might interfere with the apoptotic function of c-myc without affecting its ability to transform cells. We have not detected any significant difference in steady state levels of max RNA or protein between AR27 and parental RMN8 cells (data not shown). However, our studies do not eliminate the possibility of a qualitative change in Max which might alter the ability of the Myc/Max heterodimer to promote apoptosis. We have shown that the apoptosis-sensitive phenotype was restored in somatic cell hybrids generated between AR27 and either nontransformed or c-myctransformed Rat1A cells. We consider it unlikely that restoration of serum deprivation-induced cell death resulted from an artifact of cell fusion or an increase in gene product dosage (e.g., c-myc), given the observation that AR27 self-hybrids retained the apoptosis-resistant phenotype. Our results are therefore compatible with complementation of a recessive (i.e., loss of function) mutation, leading to abrogation of myc-mediated cell death. It is interesting to speculate that Rat1A cells, which have a pseudodiploid karyotype [Dhanaraj and Small, unpublished results], might be functionally hemizygous at the genetic locus responsible for the apoptosis-resistant phenotype. The reduction in activity or dosage of a mediator of cell death, resulting from mutation of one allele, might also diminish the ability of cells to undergo apoptosis. Alternatively, a dominant negative mutation would be compatible with the recessive behavior of this phenotype in cell hybrids. We have considered the possibility that a dominant-acting gene might be lost in intraspecies hybrids, due to the expected random segregation of chromosomes. However, there is no reason a priori to predict that such a gene would be preferentially deleted in several independently derived hybrid cell lines. Our results from somatic cell hybrid analysis suggest that activation of a dominant-acting suppressor of cell death can be excluded as being responsible for the apoptosis-resistant phenotype of AR27. In this context, we have been unable to detect expression of rat bcl2 in AR27 (or its parental cell line) by Northern or Western blot analysis (data not shown). This finding would tend to rule out the involvement of Bcl2-related proteins such as Bax or Bcl-xS, which are thought to act as negative regulators of Bcl-2 [44]. Furthermore, it is unlikely that an alteration in growth factor signaling (e.g., ectopic growth factor production, constitutive activation of a growth factor receptor or downstream effector) could account for a recessive phenotype. We have exam-
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FIG. 8. Immunoblot analysis of p53. Total cell extracts from RMN8 (lanes 1–3) and AR27 (lanes 4–6) cells were immunoprecipitated with the following anti-p53 monoclonal antibodies: mutantspecific pAb 240 (lanes 1 and 4), wild-type specific pAb 246 (lanes 2 and 5) and pan-specific antibody pAb 421 (lanes 3 and 6). Immunoprecipitates were resolved by SDS–PAGE, transferred to nitrocellulose, and assayed for p53 using PAb 421 and ECL detection.
ined whether AR27-conditioned medium, or cocultivation with AR27 cells in serum-free medium, protects parental RMN8 cells from apoptosis, and have found no evidence for any such effect (data not shown). We have determined that AR27 cells, selected for resistance to growth factor withdrawal-induced cell death, also exhibit resistance to induction of apoptosis by the general protein kinase inhibitor staurosporine. At the highest concentration of staurosporine tested, we do observe a significant amount of cell death, inferring that the apoptotic machinery is at least partially functional. Nevertheless, these results suggest that the apoptosis-resistant phenotype may be due to a mutation in a common component of the cell death pathway. There are a number of possible candidates for such a component, including interleukin-1b converting enzyme (ICE)-like cysteine proteases [45], ornithine decarboxylase [46], cyclin A [47, 48], and the p53 tumor suppressor. The wild-type p53 gene product, a positive effector of cell death in a variety of cell types [40], has recently been implicated in the myc-mediated apoptotic pathway [11, 39]. Furthermore, mutation of p53 is associated with decreased cell death from cytotoxic drugs and radiation [49]. We have examined the status of p53 in AR27 and shown it to be wild-type, on the basis of results obtained using conformation-specific antibodies to distinguish wild-type from mutant species. Within the limitations of this approach, we believe that mutation of p53 itself is unlikely to account for the resistant phenotype. In addition, we have seen no evidence for accumulation of p53 in AR27, a characteristic associated with many mutant p53 proteins [41]. We recognize, however, that wild-type p53 conformation is not necessarily synonymous with normal p53 function. Altered subcellular localization (i.e., cytoplasmic rather than nuclear), as well as loss of transcriptional repression activity [28], might be expected to result in defective p53 function with regard to apoptosis. At a minimum, our studies indicate that the point at
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which cell death is inhibited in AR27 must lie downstream of the pathway by which c-myc affects cell proliferation. Further support for this interpretation comes from a complementary genetic approach [Dhanaraj, Marcus, and Small, manuscript in preparation] in which we have isolated morphological revertants of c-myc-transformed Rat1A cells. In every case examined, selection for loss of the transformed phenotype was accompanied by loss of growth factor deprivationinduced cell death. However, it remains to be established whether c-myc mediates these disparate cellular outcomes through overlapping or independent components. It is intriguing to speculate that c-myc differentially promotes mitogenesis and apoptosis by modulating the expression of distinct target genes. Further characterization of AR27 should help to elucidate the mechanism by which c-myc promotes cell death and the role of this process in tumorigenesis. In addition, this cell line will be useful in studying the relationship between development of resistance to apoptosis and physiological processes associated with cell death, including regulation of intracellular calcium [50] and pH [51]. Finally, it has been shown that a variety of agents currently employed for cancer chemotherapy kill tumor cells by inducing apoptosis [52]. In conjunction with other established mechanisms, abrogation of apoptosis has been implicated in the development of multiple antineoplastic drug resistance [53], a significant concern in cancer therapy. We believe that AR27 cells provide an important model system to gain additional insight into this problem. We thank N. Alwis, M. Smela, and Z. Garcia for their technical assistance, and Drs. H.L. Ozer and R. Wieder for their helpful discussions and review of the manuscript. This work was supported by grants awarded to M.B.S. from the National Cancer Institute (NCI) of the National Institutes of Health (NIH, CA53136) and the Walter and Louise Sutcliffe Foundation. S.N.D. is a recipient of a fellowship from the New Jersey Commission on Cancer Research.
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Received October 16, 1995 Revised version received January 19, 1996
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AP: Exp Cell