MOLECULAR AND CELLULAR BIOLOGY, Nov. 1995, p. 5849–5857 0270-7306/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 15, No. 11
Evidence for a G2 Checkpoint in p53-Independent Apoptosis Induction by X-Irradiation ZHIYONG HAN,1 DEVASIS CHATTERJEE,1 DONG MING HE,1 JANET EARLY,2 PANAYOTIS PANTAZIS,2 JAMES H. WYCHE,1 AND ERIC A. HENDRICKSON1* Department of Molecular Biology, Cell Biology, and Biochemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912,1 and The Stehlin Foundation for Cancer Research at St. Joseph Hospital, Houston, Texas 770032 Received 10 February 1995/Returned for modification 6 April 1995/Accepted 3 August 1995
The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradiation. We demonstrate that the decision to undergo apoptosis in this cell line appears to be made at a G2 checkpoint. In addition, we characterize an HL-60 variant, HCW-2, which is radioresistant. HCW-2 cells display DNA damage induction and repair capabilities identical to those of the parental HL-60 cell line. Thus, the difference between the two cell lines appears to be that X-irradiation induces apoptosis in HL-60, but not in HCW-2, cells. Paradoxically, HCW-2 cells display high levels of expression of bax, which enhances apoptosis, and no longer express bcl-2, which blocks apoptosis. HCW-2 cells’ resistance to apoptosis may be due to the acquisition of expression of bcl-xL, a bcl-2-related inhibitor of apoptosis. In summary, apoptosis can be induced in X-irradiated HL-60 cells by a p53-independent mechanism at a G2 checkpoint, despite the presence of endogenous bcl-2. The resistance shown by HCW-2 cells suggests that bcl-xL can block this process. Exposure of cells to ionizing radiation induces the production of free oxygen and hydroxyl radicals, which in turn cause oxidative chemical damage to many constituent molecules, including lipids, proteins, and nucleic acids. Lesions in DNA such as strand scissions, single-stranded breaks, doublestranded breaks (DSBs), and base cross-links within the DNA are easily induced by ionizing radiation (12, 61). If these lesions are unrepaired or misrepaired, they can result in mutations, chromosomal aberrations, and deletions (for recent reviews, see references 23 and 45). In particular, DNA DSBs appear to be the predominant cytotoxic lesions, as even a single unrepaired DNA DSB can be a lethal event (30, 46). One additional mechanism by which ionizing radiation induces cell death is by activation of apoptosis (37, 51), a genetically determined pathway of cellular suicide required for normal development and differentiation (18, 47). The tumor suppressor protein p53 appears to be required for induction of apoptosis by ionizing radiation (for recent reviews, see references 15 and 34). Numerous studies have shown that there is a rapid accumulation of p53 in nuclei of cells following treatment with ionizing radiation and DNA-damaging chemicals (29, 39). In addition, tumor cells with mutated p53 alleles are often radioresistant (20). Impressively, it has been demonstrated that cells isolated from p53 knockout mice are resistant to apoptosis induction by X-ray irradiation (8, 38, 39). Interestingly, although X-irradiation was unable to induce apoptosis, apoptosis was induced by chemicals, such as glucocorticoids (8, 39). Thus, apoptosis induction by ionizing radiation is specifically p53 dependent. The role of p53 in the cellular response to DNA damage is thought to involve cell cycle checkpoints. Proliferating cells with a wild-type p53 allele(s) arrest at G1 following X-irradi-
ation, and this has been related to the existence of a checkpoint at which DNA damage is repaired (15, 40). It is well understood that the G1 cell cycle checkpoint is dependent upon the p53 gene exerting its effect by activating the expression of p21, an inhibitor of cyclin-dependent kinases (reviewed in reference 21). Thus, it has been postulated that p53 may control the decision of a cell to either repair DNA damage and resume proliferation or to undergo apoptosis at G1 (33). However, X-irradiated cells also arrest at the G2 phase of the cell cycle, and this checkpoint appears to be independent of p53 (32). Cells containing mutated or null p53 alleles do not arrest at G1 but still arrest at G2 (29, 32). The mechanism of mammalian G2 arrest is poorly defined. Yeast cells with a defect in the G2 checkpoint (rad9 mutants) are very sensitive to X-irradiation (63). A mammalian homolog to rad9 has not yet been described, but presumably such a gene exists and exerts a similar effect. Thus, mammalian cells exhibit G1 and G2 checkpoints, and these may control a cell’s response to DNA damage as well as its susceptibility to apoptosis. Another gene product that is known to play a role in apoptosis, but opposite from that of p53, is that of the protooncogene bcl-2 (for a recent review, see reference 31). In particular, the presence of bcl-2 can block the induction of apoptosis by many different stimuli, including ionizing radiation (52, 55). Presently, exactly how bcl-2 provides cells with radioresistance is largely unknown. It was recently demonstrated that bcl-2 can block the conversion of free oxygen radicals into peroxide and thus behaves as an antioxidant in cells (19, 28). It was speculated that bcl-2 might act as a radical scavenger that traps free radicals, thus reducing the level of active free radicals and minimizing oxidative damage to cellular macromolecules. Thus, the ability of bcl-2 to block apoptosis induction by ionizing radiation is thought to be due in large part to its role in decreasing the level of intracellular reactive oxygen species. The identification of the bcl-2 gene provided the basis for
* Corresponding author. Phone: (401) 863-3667. Fax: (401) 8632421. Electronic mail address:
[email protected]. 5849
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the isolation of numerous bcl-2-related genes, including bax (42) and bcl-x (3). Because of a differential splicing mechanism, bcl-x was found to encode bcl-xL, an apoptosis suppressor, and bcl-xS, a dominant inhibitor of bcl-2 (3). Molecular and biochemical analyses indicated that both bcl-2 and bcl-xL proteins can heterodimerize with bax or bcl-xS protein (42, 49, 66). The heterodimerization between bcl-2 or bcl-xL and bax proteins can neutralize the cell death effector activity of bax (42, 49, 66). Alternatively, bcl-xS protein can heterodimerize with bcl-2, freeing bax from bcl-2 control and consequently activating the cell death effector activity of bax (49). Together, this information suggests that the ability of a given cell to either survive or die, when an apoptotic stimulus is received, is determined by the ratio of heterodimeric apoptosis suppressors and cell death effector molecules (3, 41, 42, 49, 66). The precise interaction and hierarchy between these cell death regulatory molecules remain to be elucidated. Cells from the human promyelocytic leukemia HL-60 cell line are known to be deficient in p53 because of large deletions in the p53 gene (64). In addition, bcl-2 is expressed in HL-60 cells (26). Such a genetic background (p532/bcl-21) would predict that HL-60 cells should be resistant to apoptosis induction by ionizing radiation. However, we show here that apoptosis in HL-60 cells can be induced by X rays, thus providing an exception to the rule that p53 is required for DNA damageinduced apoptosis. Interestingly, the decision to undergo apoptosis appears to be made at a G2 checkpoint. In addition, we describe a variant, HCW-2, of the HL-60 cell line that is significantly more resistant to apoptosis induction by X rays. Importantly, we show that the resistance to X-irradiation by HCW-2 cells occurs in the absence of bcl-2 and instead correlates with the acquisition of expression of bcl-xL. Thus, HCW-2 cells should prove to be a very useful tool for dissecting the roles of p53, bcl-2, and bcl-xL in apoptosis.
MOL. CELL. BIOL. into a 1.0% horizontal agarose gel in 0.53 TBE (45 mM Tris [pH 8.2], 45 mM boric acid, 1.25 mM EDTA) with 0.5 mg of ethidium bromide per ml and subjected to PFGE for 28 h in a homemade (50) pulsed-field gel chamber at 108C and 150 V. Under these electrophoresis conditions, chromosomal DNA which had broken into 3- to 5-Mbp fragments migrated as a discrete band on the gel. Gel slices corresponding to the DNA in the band and DNA remaining in the well were excised from the gel, and the 3H counts per minute in each slice were determined. The percentage of DNA released corresponds to the radioactivity in the band divided by the total radioactivity. Northern (RNA) analyses. Total RNA was isolated by the method of Gough (11), and 50 mg was electrophoresed through a 1.2% agarose gel containing 6% formaldehyde. The RNA was transferred to nitrocellulose filters and immobilized via UV irradiation (Stratalinker; Stratagene, La Jolla, Calif.). The filters were incubated in 50% formamide–53 Denhardt’s solution–0.5% sodium dodecyl sulfate (SDS)–100 mg of salmon sperm DNA per ml at 428C for 4 h and were then incubated in 50% formamide–53 Denhardt’s solution–53 SSPE (13 SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7])–0.1% SDS–100 mg of salmon sperm DNA per ml–106 cpm of radiolabeled probe per ml for 15 h at the same temperature. The filters were washed three times in 0.13 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate) buffer containing 0.1% SDS at 608C with a change every 45 min. Afterwards, the filters were dried in the air and exposed to X-ray films at 2838C. DNA probes were radiolabeled with [a-32P]dATP by using random priming reagents purchased from Stratagene. The probe utilized for the bcl-xL Northern analysis was a 40-mer oligonucleotide (59-CCTGGATCCAAGGCTCTAGGTGGTCATTCAGGTAAGTGG C-39) which specifically recognizes bcl-xL (6). Radiolabeling of the probe and the filter hybridization and washing conditions were precisely as described previously (13). Western analyses. A total of 107 cells were washed twice in PBS and then lysed in 200 ml of 1 mM EDTA—0.2% Triton X-100—10 mg of leupeptin per ml–10 mg of E64 per ml–10 mg of pepstatin per ml–1 mg of aprotinin per ml–20 mg of chymostatin per ml–50 mg of bestatin per ml–50 mg of phosphoramidon per ml–50 mg of antipan-dihydrochloride per ml. The lysate was centrifuged at 12,500 3 g for 10 min at 48C. The supernatant was recovered and mixed with 5 volumes of acetone. The mixture was incubated at 2208C for 5 h to precipitate proteins. The proteins were pelleted by centrifugation, resuspended in 100 ml of SDSpolyacrylamide gel electrophoresis sample loading buffer, and heated at 1008C for 5 min. One-fifth of the sample was then electrophoresed in a 10% polyacrylamide gel under nonreducing conditions. Afterwards, the proteins were transferred to nitrocellulose via electrophoresis in a Trans-Blot chamber (Bio-Rad) and developed with ECL-Western blot reagents (Amersham) according to the protocols provided by the manufacturers. Flow cytometry determinations. The flow cytometry procedure has been described in detail elsewhere (43).
MATERIALS AND METHODS Materials. Fetal bovine serum was purchased from Sigma Chemical Co. (St. Louis, Mo.). Tritiated thymidine was purchased from Du Pont-NEN, Inc. (Boston, Mass.). Monoclonal antibodies against human bcl-2 and DNA probes for the human p53, retinoblastoma (Rb), and bcl-2 genes were purchased from Oncogene Science, Inc. (Uniondale, N.Y.). Polyclonal antibody against human bax protein was obtained from Santa Cruz Biotechnology, Inc. cDNAs encoding human bcl-xL and bcl-xS were kindly provided by Craig B. Thompson (Howard Hughes Medical Institute, Departments of Medicine and Molecular Genetics and Cell Biology, University of Chicago, Chicago, Ill.). Enhanced chemiluminescence (ECL)-Western blot (immunoblot) analysis reagents were purchased from Amersham Life Science, Inc. (Arlington Heights, Ill.). Protease inhibitors were purchased from Boehringer Mannheim Co. (Indianapolis, Ind.). Cells. HL-60 cells were purchased from American Type Culture Collection (Rockville, Md.). HCW-2 cells were isolated as described elsewhere (13). All cells were cultured in RPMI 1640 medium (Gibco BRL) supplemented with 20% fetal bovine serum, 100 U of penicillin per ml, and 50 U of streptomycin per ml. Cell cultures were kept in a humidified incubator with 5% CO2 at 378C. Determination of cell survival by growth assays. Exponentially growing cells were X-irradiated by using either a Philips 250-kV-peak X-ray machine at a dose rate of 2.2 Gy/min with a 0.35-mm-thick copper filter or a 137Cs source at a dose rate of 11.1 Gy/min. Cells were then plated in 96-well plates by limiting dilution at densities ranging from 0.5 to 5,000 cells per well and incubated for 2 to 3 weeks (16). At the end of incubation, wells containing a cell lawn were scored. Survival of X-irradiated cells was compared with that of cells which had not been irradiated. DNA fragmentation assay. The DNA fragmentation assay has been described in detail previously (14). PFGE analyses of DNA DSBs. Cells were cultured in medium containing [3H]thymidine (0.02 mCi/ml; 80 Ci/mmol) for 24 h and then subjected to a chase period of 24 h to uniformly radiolabel the chromosomal DNA (16, 36). The cells were then X-irradiated with a 137Cs source at a dose rate of 11.1 Gy/min. To prepare cells for pulsed-field gel electrophoresis (PFGE) analyses, cells were washed in phosphate-buffered saline (PBS) and resuspended at 107/ml in 1.0% low-melting-point agarose at 428C (1). The cell-agarose slurry was then cast into agarose plugs (80 ml), which were subsequently lysed in proteinase K, washed, and treated with RNase A as described previously (1). The plugs were then cast
RESULTS Mutant isolation. We have isolated a variant (13), HCW-2, of the human promyelocytic leukemia HL-60 cell line which is highly resistant to the cytotoxic effects of the chemotherapeutic compound 8-Cl-cyclic AMP (7). This characteristic and other attributes of the HCW-2 cell line are described elsewhere (13). In the course of testing HCW-2 with other chemotherapeutic regimens, however, we observed that the HCW-2 cell line was also extremely radioresistant and apoptosis resistant. These results are described below. HL-60 cells are X ray sensitive, whereas HCW-2 cells are radioresistant. Numerous studies have shown that p53-deficient cells (8, 38, 39) or cells with mutated p53 alleles (20) are radioresistant. Since HL-60 cells lack p53 (64), we were surprised to find that the survival of HL-60 cells was sensitive to X-irradiation. HL-60 cells were X-irradiated (0 to 10 Gy), and the ability of cells to survive and form colonies 2 weeks postirradiation was scored (Fig. 1). The irradiation dose required to reduce cell survival to 37% (D37) is an index used to compare X-ray sensitivities (16). The D37 for HL-60 was 1.5 Gy, which is a value expected for a mammalian hematopoietic cell lineage with a wild-type p53 allele (2, 16, 17, 58). Thus, HL-60 cells do not exhibit the radioresistant phenotype expected for a p53deficient cell line. HCW-2 cells were found to be much more resistant to the lethal effect of X rays than were HL-60 cells (Fig. 1). The difference in survival between the two cell lines was observed at all doses examined, including very low doses (e.g., 1.5 Gy, Fig. 1). The D37 for HCW-2 cells was 2.8 Gy, a significant 1.9-fold
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FIG. 1. HL-60 cells are X-ray sensitive, whereas HCW-2 cells are radioresistant. HCW-2 and HL-60 cells were exposed to the indicated doses of X rays and plated under limiting dilution, and then approximately 2 weeks postirradiation the fraction of cells surviving to form colonies was scored. The profiles are the averages of two experiments, each performed in duplicate.
increase over that of the parental HL-60 cell line. Thus, HCW-2 mutant cells are radioresistant and exhibit an X-rayresponsive phenotype more similar to that of other p53-deficient cells. HL-60 and HCW-2 cells are equally sensitive to the induction of DNA DSBs by X rays. The first possibility we considered was whether the radiosensitivities of these cell lines could be explained by X rays inducing more DNA damage in HL-60 cells than in HCW-2 cells. Therefore, using DNA DSB induction by X rays as a parameter, we assessed the radiosensitivities of HL-60 and HCW-2 cells at the biochemical level. We first performed a dose-response experiment using PFGE to quantitate the number of DSBs in chromosomal DNA following X-ray exposure without DNA repair (1, 2, 36). Under our electrophoresis conditions, intact genomic DNA remains in the gel well, whereas smaller (X-ray-broken) DNA migrates into the gel as a tight smear that appears as a band (36, 54). Importantly, the amount of DNA released from the well is directly proportional to the number of unrepaired DSBs in the cell (1, 54). Exponentially growing cultures of each cell line were irradiated with increasing doses of X rays and immediately processed for PFGE so that no repair could take place (see Materials and Methods). This experiment therefore measured only the amount of initial DNA DSBs in the cells fol-
lowing X irradiation. All of the cell lines showed almost identical linear responses to X-irradiation, with increasing amounts of DNA released with increasing X-ray doses (Fig. 2). Thus, the radiosensitivity of HL-60 cells could not be explained by a greater level of initial X-ray-induced DNA damage. HL-60 and HCW-2 cells repair DNA DSBs with equivalent efficiencies. The above results indicated that for a given X-ray dose, equivalent numbers of DNA DSBs were introduced into HL-60 and HCW-2 cells. However, if HL-60 cells were not as efficient in repairing DNA DSBs as HCW-2 cells, then the unrepaired lesions could potentially explain the radiosensitivity of HL-60 cells. To investigate this possibility, HL-60 and HCW-2 cells were X-irradiated at 80 Gy and placed at 378C. At various intervals during the subsequent 2 h, cells were assessed for the extent of DNA DSB repair which had taken place by using the PFGE assay (see Materials and Methods). The results showed that both HL-60 and HCW-2 cells were able to repair most (70%) DNA DSBs within 2 h after X-irradiation (Fig. 3A). Furthermore, HL-60 and HCW-2 cells repaired DNA DSBs with almost identical kinetics and efficiency (Fig. 3B). Thus, the radiosensitivity of HL-60 cells was not due to a reduced ability to repair DNA DSBs. HL-60 and HCW-2 cells exhibit a G2, but not a G1, checkpoint. We next determined if regulatory cell cycle checkpoints
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FIG. 2. The levels of DNA DSB induction in HL-60 and HCW-2 cells following X-irradiation are identical. (A) PFGE analysis of HL-60 and HCW-2 chromosomal DNA following X-irradiation. HL-60 and HCW-2 cells were radiolabeled with [3H]thymidine, exposed to the indicated doses of X-irradiation (in grays), and then immediately processed for PFGE as described in Materials and Methods. Under these conditions, damaged DNA migrates out of the well as a discrete band. (B) DNA DSB induction is identical in HL-60 and HCW-2 cells. Gel slices corresponding to the well and the bands of each lane of the gel shown in panel A were individually excised, and the amount of 3H (counts per minute) in each slice was determined. The data are presented as percentages of 3H counts per minute in the band (percent DNA released) versus increasing X-ray dose and are the averages of two experiments, each performed in duplicate.
were normal in HL-60 and HCW-2 cells. To address whether HL-60 and HCW-2 cells behaved as p53-null cell lines, asynchronously growing cells were X-irradiated and the fractions of cells in various phases of the cell cycle were quantitated by flow cytometry at various times postirradiation. The data clearly showed that, as expected, neither cell line showed a significant G1 arrest checkpoint (Fig. 4A, histograms d and i). Both cell lines showed a strong G2 checkpoint arrest, with .90% of the cells from each cell line arrested in G2 by 20 h postirradiation (Fig. 4A, histograms e and j). Thus, the initial cell cycle responses to DNA damage of these two cell lines appeared to be normal (for p53null cell lines) and indistinguishable.
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FIG. 3. The capacities for DNA DSB repair in HL-60 and HCW-2 cells following X-irradiation are identical. (A) PFGE analysis of HL-60 and HCW-2 chromosomal DNA following X-irradiation. Aliquots of HL-60 and HCW-2 cells were radiolabeled with [3H]thymidine and were either left untreated (U) or exposed to 80 Gy of X rays and then incubated at 378C for the indicated times (in minutes) before being processed for PFGE as described in Materials and Methods. (B) Identical DNA DSB repair in HL-60 and HCW-2 cells. Gel slices corresponding to the well and the bands of each lane of the gel shown in panel A were individually excised, and the amount of 3H (counts per minute) in each slice was determined. The data are presented as percentages of 3H counts per minute remaining in the band (percent DNA repair) versus increasing time and are the averages of two experiments, each performed in duplicate.
HCW-2 cells recover from G2 arrest and continue cycling, whereas HL-60 cells undergo apoptosis. When the above experiment was extended for longer intervals, however, a difference between HL-60 and HCW-2 cells became obvious. At 24 h after X-irradiation, approximately 25% of the HCW-2 cells had moved from G2 into G1 (Fig. 4B, histogram f). At even later times, the HCW-2 cells exhibited a profile virtually indistinguishable from that of the original asynchronously growing population of cells, suggesting that they had successfully reentered the cell cycle (Fig. 4B, histograms g and h). The small percentage of HCW-2 cells with less than a G1 amount of DNA corresponds to cells undergoing necrosis (22). In striking con-
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FIG. 4. Cell cycle arrest checkpoints in HL-60 and HCW-2 cells following X-irradiation. (A) Fluorescence-activated cell sorter (FACS) analysis of asynchronously growing HL-60 (histograms a to e) and HCW-2 (histograms f to j) cells following X-irradiation with 10 Gy demonstrates that both cell lines arrest in the G2, but not the G1, phase of the cell cycle. Cells were analyzed immediately prior to irradiation (a and f) and then at 5 (b and g), 10 (c and h), 15 (d and i), and 20 (e and j) h postirradiation. (B) FACS analysis of asynchronously growing HL-60 (histograms a to d) and HCW-2 (histograms e to h) cells following X-irradiation with 10 Gy demonstrates that HCW-2 cells recover from growth arrest and resume cycling, whereas HL-60 cells initiate apoptosis (Ap). Cells were analyzed immediately prior to irradiation (a and e) and then at 24 (b and f), 48 (c and g), and 72 (d and h) h postirradiation. Nc, necrotic cells.
trast, HL-60 cells never recovered from the G2 checkpoint arrest. At 24 h after X-irradiation, .90% of the HL-60 cells still remained in G2 (Fig. 4B, histogram b). At later times, a large fraction of HL-60 cells with less than a G1 amount of DNA appeared, which corresponds to cells undergoing apoptosis (13, 22) (Fig. 4B, histograms c and d). Thus, following DNA damage, HCW-2 cells appeared to recover from the G2 cell cycle arrest and resumed cycling, whereas the vast majority of HL-60 cells did not resume cycling but instead appeared to initiate apoptosis. X-irradiated HL-60 cells die by apoptosis. The appearance of HL-60 cells with less than a G1 amount of DNA was suggestive that the X-irradiation-induced death of HL-60 cells occurred by apoptosis (13, 22). To experimentally confirm this hypothesis, the appearance of DNA fragmentation, another cardinal feature of apoptosis (65), following X-irradiation was determined. Cells were exposed to X rays (10 Gy), and cellular DNA was subsequently isolated at 72 h postirradiation and analyzed by agarose gel electrophoresis. In HL-60 cells, there was a large amount of fragmented DNA (Fig. 5, compare lanes 1 and 2). The observed nucleosomal, ladder-like pattern of fragmented DNA is a hallmark of apoptosis (65). As expected, there was little DNA degradation in X-irradiated HCW-2 cells even at 72 h postirradiation (Fig. 5, compare lanes 3 and 4). Thus, X-irradiated HL-60 cells died by apoptosis, whereas HCW-2 cells were resistant to apoptosis induction by X rays. G1-arrested HL-60 cells are resistant to apoptotic induction by X rays. The above results strongly indicated that following DNA damage, HL-60 cells progressed to the G2 phase of the cell cycle, whereupon they initiated apoptosis. To experimentally confirm the requirement for HL-60 cells to be in G2 to undergo apoptosis, we investigated the apoptotic sensitivity of HL-60 cells arrested at G1. HL-60 cells were incubated in serum-free or serum-containing (20% fetal bovine serum) me-
dium for 24 h, and at the end of this incubation, approximately 90% of the cells in serum-free medium were arrested at G1 (Fig. 6f). These two sets of cells were X-irradiated with 10 Gy and continuously incubated in the absence or presence of serum for a further 96 h. At various intervals, the cells were analyzed for cell cycle progression and apoptosis by flow cytometry. In contrast to the cells incubated in the serum-containing medium, which underwent significant apoptosis (Fig. 6b to e), cells incubated in serum-free medium underwent only
FIG. 5. HL-60 cells undergo apoptotic DNA fragmentation following X-irradiation. HL-60 (lanes 1 and 2) and HCW-2 (lanes 3 and 4) cells were either left untreated (lanes 1 and 3) or exposed to 10 Gy of X rays (lanes 2 and 4), and at 72 h postirradiation the status of chromosomal DNA was assessed by agarose gel analysis. Lane M, 123-bp DNA markers.
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cells are deficient in p53 because of large deletions in the p53 gene (64). Thus, it was not a surprise to see that both HL-60 and HCW-2 cells lacked expression of p53 mRNA (Fig. 7A). The above unexpected results led us to examine the possibility that X-irradiation might down regulate bcl-2 expression in HL-60 cells and induce it in HCW-2 cells. However, Western analysis showed that 10 and 25 h after X-irradiation, there was neither reduction in the bcl-2 protein level in HL-60 cells (Fig. 7B, lanes 1 to 3) nor induction of its expression in HCW-2 cells (Fig. 7B, lanes 4 to 6). Thus, paradoxically, HCW-2 cells are more radioresistant than the parental HL-60 cells, even though they do not express bcl-2. Expression of bax is enhanced in HCW-2 cells. The activity of bcl-2 within cells is thought to be modulated by bax (42). In particular, bcl-2 and bax proteins form heterodimers, and it has been postulated that the ratio of bcl-2 to bax is critical in determining the susceptibility of a cell to apoptotic signals (41). Thus, we determined the status of bax in these two cell lines. Northern analysis showed that bax mRNA was approximately equally expressed in the two cell lines (Fig. 8A, compare lanes 1 and 4). X-irradiation resulted in a slight down regulation of bax mRNA in HL-60 cells at 24 and 48 h (Fig. 8A, lanes 2 and 3), while bax expression remained unchanged in HCW-2 cells (Fig. 8A, lanes 5 and 6). Interestingly, Western analysis showed that, in comparison with HL-60 cells, bax was highly overexpressed in HCW-2 cells (Fig. 8B, compare lanes 1 and 4). This observation suggests that significant regulation of bax may occur at the posttranscriptional level (13). In HL-60 cells, bax protein increased slightly following X-irradiation in a temporal manner that correlated well with the onset of apoptosis in
FIG. 6. G1 arrest of HL-60 cells prevents apoptosis. HL-60 cells were incubated in serum-containing (a) or serum-free (f) medium for 24 h and then analyzed by flow cytometry. Cells were also X-irradiated (10 Gy) and incubated in the presence (b to e) and absence (g to j) of serum for an additional 24 (b and g), 48 (c and h), 72 (d and i), and 96 (e and j) h. The cells were then analyzed by flow cytometry for apoptosis (Ap).
very limited apoptosis (Fig. 6g to j). Since a fraction of the HL-60 cells in the serum-free medium did eventually progress to G2 (Fig. 6g to j), these may be the cells which eventually underwent apoptosis. Thus, we conclude that most, if not all, HL-60 cells must be in G2 of the cell cycle in order to initiate apoptosis induced by X-irradiation. HCW-2 cells are deficient in the expression of the bcl-2 gene. Apoptosis induced by a variety of stimuli, including X-irradiation, has been shown to be abrogated by bcl-2 in many different cell types (52, 55). Therefore, we anticipated that one explanation for the radioresistance and apoptosis resistance of the HCW-2 cell line might be due to a mutation that caused activation or overexpression of bcl-2 in these cells in comparison with the expression in the parental HL-60 cells. Northern blot analysis, however, indicated that there were already high levels of bcl-2 mRNA in HL-60 cells (Fig. 7A), in agreement with previous observations (26). Even more surprising was the finding that there was no detectable bcl-2 mRNA in HCW-2 cells (Fig. 7A). When the filter was reprobed for the expression of the Rb gene, it was observed that HL-60 and HCW-2 cells expressed similar amounts of Rb mRNA (Fig. 7A). Therefore, the lack of detectable bcl-2 mRNA in HCW-2 cells was not due to a loading error during gel electrophoresis or the subsequent transfer process. It has been demonstrated previously that HL-60
FIG. 7. HCW-2 cells do not express bcl-2. (A) Northern blot analysis of HL-60 and HCW-2 cells. Total RNA was prepared from HL-60 (lane 1) and HCW-2 (lane 2) cells, electrophoresed through an agarose gel, transferred to nitrocellulose, and subsequently hybridized with probes specific for the bcl-2, p53, and Rb genes. (B) Western analysis of bcl-2 expression. HL-60 (lanes 1 to 3) and HCW-2 (lanes 4 to 6) cells were exposed to 10 Gy of X rays, and then at 0 (lanes 1 and 4), 10 (lanes 2 and 5), or 25 (lanes 3 and 6) h, cell extracts were prepared and subjected to a Western analysis using an antibody specific for bcl-2.
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(5). Thus, the apoptotic resistance exhibited by HCW-2 cells may be due to the acquisition of bcl-xL expression. DISCUSSION
FIG. 8. HCW-2 cells overexpress bax, but the pattern of gene expression does not correlate with X-irradiation. HL-60 (lanes 1 to 3) and HCW-2 (lanes 4 to 6) cells were exposed to 10 Gy of X rays, and then at 0 (lanes 1 and 4), 24 (lanes 2 and 5), and 48 (lanes 3 and 6) h, cell extracts were prepared and subjected to Northern analysis (A) or Western analysis (B) using probes specific for bax.
these cells (Fig. 8B, lanes 2 and 3). However, in HCW-2 cells, bax expression was unaffected by X-irradiation at subsequent times (Fig. 8B, lanes 5 and 6). Thus, again paradoxically, HCW-2 cells are more radioresistant and apoptosis resistant than the parental HL-60 cells, even though they overexpress bax. The level of expression of bcl-x in HCW-2 cells is elevated. The pattern of expression of bcl-2 and bax in HCW-2 cells was inconsistent with the proposed roles of these genes in regulating apoptosis, suggesting that the DNA damage-induced response in this cell line might be mediated by other apoptotic regulators. The bcl-x gene has been demonstrated to encode both an apoptotic activator, bcl-xS, and an apoptotic suppressor, bcl-xL (3). Importantly, it has been demonstrated that bcl-xL can suppress apoptosis in certain situations in which bcl-2 is ineffective (10). Northern analysis demonstrated that bcl-xL was not expressed in HL-60 cells, nor was it induced upon X-irradiation (Fig. 9). In contrast, bcl-xL was expressed at high levels in HCW-2 cells, though this expression was not induced 24 h after X-irradiation (Fig. 9). The lack of induction of bcl-xL following DNA damage has been previously observed
FIG. 9. Expression of the bcl-xL gene in HCW-2 cells. HL-60 and HCW-2 cells were either not X-irradiated (2 lanes) or X-irradiated (1 lanes) with 10 Gy and then incubated for 24 h. Total RNA was isolated from the cells, and the expression of bcl-xL mRNA was detected by Northern analysis using an oligonucleotide specific for bcl-xL.
In this report, we document that the p53-deficient cell line HL-60 can undergo apoptosis following X-irradiation, demonstrating that the p53 gene product is not necessary for this process. In addition, we describe the characterization of an HL-60 variant cell line, HCW-2, which is radioresistant and apoptosis resistant. We demonstrate that the radioresistant properties of HCW-2 are independent of the proto-oncogene bcl-2. Importantly, we show that there is no difference between these two cell lines in either the number of DNA DSBs introduced into chromosomal DNA following X-irradiation or the repair of those DSBs. These results have important implications for the possible roles of the p53 gene and bcl-2 in mediating DNA damage-induced apoptosis. p53-independent apoptosis induction by X rays. A number of studies have shown that apoptosis induction by X rays and DNA-damaging chemicals appeared to be dependent on the presence of a wild-type p53 allele (4, 8, 38, 39). It was also clear that in the absence of wild-type p53, apoptosis could still be induced by other stimuli, such as glucocorticoids and cycloheximide (8, 13, 39). Thus, wild-type p53 was thought to be uniquely required for apoptosis induction by ionizing radiation and DNA-damaging chemicals. Our results clearly show, however, that apoptosis initiated by DNA damage can occur in a p53-independent fashion. Recently, Strasser et al. (56) reported that activated T cells and T-cell lymphoma cell lines derived from p532/p532 mice were sensitive to X-irradiation and other agents that cause DNA damage. Thus, in at least two different cell types, HL-60 cells and T lymphocytes, there exists a p53-independent pathway in which DNA damage can still trigger apoptosis. Such a p53-independent pathway may exist in many cell types. What is the role of p53 in apoptosis? A recent study discovered that UV irradiation of somatotropic progenitor cells induced apoptosis in a p53-dependent fashion (4). This was shown to occur in the presence of either RNA or protein synthesis inhibitors and consequently in the absence of expression of p53-regulated genes. Thus, it was hypothesized that wild-type p53 might be directly involved in either the repair of DNA lesions or cleavage of apoptotic DNA in cells (4). Our data do not support either one of these models. First, we demonstrated that the extents of DNA DSB induction by X rays in HL-60 and HCW-2 cells (Fig. 2) were identical, ruling out the possibility that exposure of p53-deficient cells to X rays simply does not cause damage to DNA. Second, we have shown that extensive DNA repair occurs in both HL-60 and HCW-2 cells following X-irradiation (Fig. 3), even though both of these cell lines are p53 deficient. The level of DNA repair observed in these cell lines was very similar to that observed in large variety of p53 wild-type cell lines (1, 2, 16). In addition, no DNA repair defect was observed following UV irradiation of a p53-deficient mouse cell line (24). Thus, a direct role for p53 in DNA repair seems to be ruled out. We have also demonstrated that X rays can induce apoptosis in the p53deficient HL-60 cell line (Fig. 4 and 5). This agrees with the observation that apoptosis can still be stimulated by other factors, such as glucocorticoids, in p53-knockout animals (8, 39). Thus, it also seems highly unlikely that p53 actively participates in apoptotic DNA cleavages. Alternatively, we favor a model in which p53 acts as an activator of apoptosis when DNA is damaged. The very recent observation that p53 can bind to damaged DNA is consistent
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with this model (27, 35). Since several studies have suggested that transcription may not be required for apoptosis (4, 13, 59, 62), the ability of p53 to activate apoptosis may be independent of its ability to act as a transcription factor (reviewed in reference 60). However, since p53 is known to be capable of physically associating with a plethora of cellular proteins (reviewed in reference 44), it is plausible that p53 may exert an apoptotic regulatory function through one of these factors, independent of transcription. In any event, we predict that p53 acts far upstream in the apoptotic pathway so that other genes are likely to be epistatic to p53. The expression of these genes presumably explains why HL-60 and other p53-deficient cell lines can still undergo apoptosis. Evidence for a G2 checkpoint that regulates apoptosis in the absence of p53. As expected for p53-deficient cell lines (29), both HL-60 and HCW-2 cells lacked a G1 checkpoint following X-irradiation (Fig. 4A). We saw no evidence for a p53-independent G1 checkpoint, as has been observed recently for some activated T cells and T-lymphoma cells (56). Our data indicated that X-irradiated HL-60 and HCW-2 cells reached the G2 phase of the cell cycle at about the same time and arrested normally (Fig. 4A). Interestingly, after arresting, HCW-2 cells eventually resumed cycling, whereas HL-60 cells appeared to never leave G2 but instead initiated apoptosis (Fig. 4B). Therefore, these results suggest that there is a critical factor active during the G2 checkpoint which regulates apoptosis. Such a suggestion was also proposed in a study using p53-deficient proliferating T cells (56). Together, these results suggest that a G2 checkpoint factor monitors the integrity of DNA so that when DNA damage is unrepaired or misrepaired, it signals cells to arrest at this point and initiate apoptosis. In the case of HCW-2, this putative G2 checkpoint factor may be suppressed by bcl-xL, allowing these cells to go through mitosis and survive. However, survival would not come without a cost, and this model predicts that the mutation rate for HCW-2 cells is higher than that for HL-60 cells. We are currently testing this hypothesis. A recent report demonstrated that X-ray-irradiated Baf-3 cells which failed to arrest in G1 but instead entered S phase underwent rapid apoptotic cell death, whereas Baf-3 cells arrested in G1 did not undergo apoptosis (5). These results suggested that progression out of G1 is required in order for DNA damage-induced apoptosis to occur. Similarly, in contrast to cycling HL-60 cells, HL-60 cells that were arrested in G1 and subsequently X-irradiated were resistant to apoptosis (Fig. 6). However, our results are more consistent with an additional requirement for progression into G2 and not just progression into S phase, as we saw no evidence for the induction of apoptosis in S phase (Fig. 6). Role of bcl-2 in ameliorating X-ray-induced apoptosis. bcl-2 has emerged as the most significant biologically relevant apoptosis suppressor (reviewed in reference 31). Induction of apoptosis in many cell types by a variety of stimuli, including X rays, has been demonstrated to be abrogated by bcl-2. The only documented biochemical function of the bcl-2 protein is that of an antioxidant (19, 28). Thus, it was proposed that bcl-2 might act as a scavenger to reduce intracellular levels of reactive free oxygen radicals (28). Alternatively, it was proposed that bcl-2 might block the conversion of free oxygen radicals into peroxide to prevent the oxidation of molecules such as lipids (19). However, despite intensive investigation, the precise mechanism by which bcl-2 accomplishes apoptotic suppression is unknown. The ability of bcl-2 to inhibit cell death through reduced free radical formation at first glance seems very relevant to DNA damage caused by X-irradiation. That is because the mecha-
MOL. CELL. BIOL.
nism by which X rays cause cell death is extremely well defined (9, 12). Ionizing radiation energy causes the generation of free oxygen and hydroxyl radicals in cells, which subsequently induce oxidative damage to constituent molecules, including proteins, lipid membranes, and DNA. Thus, it was hypothesized that bcl-2 bestows radioresistance because it reduces oxidative damage to various cellular macromolecules (19). In this study, we demonstrate that HL-60 cells express a high level of bcl-2 protein, whereas HCW-2 cells express none. However, we found that there was no difference between the cell lines when we used DNA DSB induction as a biochemical parameter for assessing the oxidative damage to DNA by X rays (Fig. 2). Thus, the presence of Bcl-2 in HL-60 cells does not provide the cells protection against DNA strand breaks caused by free radicals. These results strongly support two very recent reports that Bcl-2 could still mediate apoptotic suppression even under anaerobic conditions, in which free radicals would not have been present (25, 53). Lastly, our results demonstrating identical repair capabilities for HL-60 and HCW-2 cells (Fig. 3) rule out an active role for bcl-2 in DNA repair. A possible role for bcl-xL in protecting HCW-2 cells from DNA damage-induced apoptosis. Regardless of the mechanism by which bcl-2 imparts radioresistance to certain cells, we have demonstrated here that HCW-2 cells are radioresistant and apoptosis resistant in the absence of bcl-2. Thus, a gene epistatic to bcl-2 appears to be altered in HCW-2 cells. Besides bcl-2, the products of several other genes, such as BAG-1 (57), mcl-1 (48), and bcl-xL (3) (for a recent review, see reference 41), have been considered as potential apoptosis suppressors. Thus, it was particularly interesting to see that, in contrast to HL-60 cells, HCW-2 cells highly expressed bcl-xL (Fig. 9). It is thus tempting to speculate that bcl-xL might protect cells at the G2 checkpoint. To experimentally test this hypothesis, we are currently introducing bcl-xL into HL-60 cells. In summary, we demonstrate that p53 is not required for all forms of DNA damage-induced apoptosis. In addition, we describe a novel bcl-2-deficient human cell variant, HCW-2, which is resistant to apoptotic induction by DNA damage, possibly because of the presence of bcl-xL. Lastly, our data address several possible roles for p53 and bcl-2 and suggest that an important checkpoint for apoptotic induction exists at G2. ACKNOWLEDGMENTS We thank John T. Leith for granting us unlimited access to his laboratory and his X-ray equipment. E.A.H. is a Leukemia Society of America Scholar. This work was supported in part by monies provided by Brown University and The Stehlin Foundation for Cancer Research. REFERENCES 1. Ager, D. D., and W. C. Dewey. 1990. Calibration of pulsed field gel electrophoresis for measurement of DNA double-strand breaks. Int. J. Radiat. Biol. 58:249–259. 2. Biedermann, K. A., J. Sun, A. J. Giaccia, L. M. Tosto, and J. M. Brown. 1991. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc. Natl. Acad. Sci. USA 88:1394–1397. 3. Boise, L. H., M. Gonzalez-Garcia, C. E. Postema, L. Ding, T. Lindsten, L. A. Turka, X. Mao, G. Nunez, and C. B. Thompson. 1993. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74:597–608. 4. Caelles, C., A. Helmberg, and M. Karin. 1994. p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature (London) 370:220–223. 5. Canman, C. E., T. M. Gilmer, S. B. Coutts, and M. B. Kastan. 1995. Growth factor modulation of p53-mediated growth arrest versus apoptosis. Genes Dev. 9:600–611. 6. Chen, M., J. Quintans, Z. Fuks, C. Thompson, D. W. Kufe, and R. R. Weichselbaum. 1995. Suppression of Bcl-2 messenger RNA production may mediate apoptosis after ionizing radiation, tumor necrosis factor a, and ceramide. Cancer Res. 55:991–994.
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7. Cho-Chung, Y. S., T. Clair, G. Tortora, and H. Yokozaki. 1991. Role of site-selective cAMP analogs in the control and reversal of malignancy. Pharmacol. Ther. 50:1–33. 8. Clarke, A. R., C. A. Purdie, D. J. Harrison, R. G. Morris, C. C. Bird, M. L. Hopper, and A. H. Wylie. 1993. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature (London) 362:849–852. 9. Goodhead, D. T., J. Thacker, and R. Cox. 1993. Effects of radiations of different qualities on cells: molecular mechanisms of damage and repair. Int. J. Radiat. Biol. 63:543–556. 10. Gottschalk, A. R., L. H. Boise, C. B. Thompson, and J. Quintans. 1994. Identification of immunosuppressant-induced apoptosis in a murine B-cell line and its prevention by bcl-x but not bcl-2. Proc. Natl. Acad. Sci. USA 91:7350–7354. 11. Gough, N. M. 1988. Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. Anal. Biochem. 173:93–95. 12. Halliwell, B., and J. M. C. Gutteridge. 1989. Free radicals in biology and medicine, 2nd ed. Clarendon Press, Oxford. 13. Han, Z., D. Chatterjee, J. Early, P. Pantazis, E. A. Hendrickson, and J. H. Wyche. Isolation and characterization of an apoptosis-resistant mutant of human leukemia HL-60 cells that has switched expression from bcl-2 to bcl-x. Submitted for publication. 14. Han, Z., and J. H. Wyche. 1994. Guanosine induces necrosis of cultured aortic endothelial cells. Am. J. Pathol. 145:423–427. 15. Hartwell, L. 1993. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 71:543–546. 16. Hendrickson, E. A., X.-Q. Qin, E. A. Bump, D. G. Schatz, M. Oettinger, and D. T. Weaver. 1991. A link between double-strand break-related repair and V(D)J recombination: the scid mutation. Proc. Natl. Acad. Sci. USA 88: 4061–4065. 17. Hendry, J. H. 1988. Survival of cells in mammalian tissues after low doses of irradiation: a short review. Int. J. Radiat. Biol. 53:89–94. 18. Hengartner, M. O., and H. R. Horvitz. 1994. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell 76:665–676. 19. Hockenbery, D. M., Z. N. Oltvai, X.-M. Yin, C. L. Milliman, and S. J. Korsmeyer. 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251. 20. Hollstein, M., D. Sidransky, B. Volgelstein, and C. C. Harris. 1991. p53 mutations in human cancers. Science 253:49–53. 21. Hunter, T. 1993. Braking the cycle. Cell 75:839–841. 22. Huschtscha, L. I., T. M. Jeitner, C. E. Andersson, W. A. Bartier, and M. H. N. Tattersall. 1994. Identification of apoptotic and necrotic human leukemic cells by flow cytometry. Exp. Cell Res. 212:161–165. 23. Hutchinson, F. 1993. Molecular biology of mutagenesis of mammalian cells by ionizing radiation. Semin. Cancer Biol. 4:85–92. 24. Ishizaki, K., Y. Ejima, T. Matsunaga, R. Hara, A. Sakamoto, M. Ikenga, Y. Ikawa, and S. Aizawa. 1994. Increased UV-induced SCEs but normal repair of DNA damage in p53-deficient mouse cells. Int. J. Cancer 58:254–257. 25. Jacobson, M. D., and M. C. Raff. 1995. Programmed cell death and Bcl-2 protection in very low oxygen. Nature (London) 374:814–816. 26. Jarvis, W. D., A. J. Turner, L. F. Povirk, R. S. Traylor, and S. Grant. 1994. Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res. 54:1707–1714. 27. Jayaraman, L., and C. Prives. 1995. Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell 81:1021–1029. 28. Kane, D. J., T. A. Sarafian, R. Anton, H. Hahn, E. B. Gralla, J. S. Valentine, T. Ord, and D. E. Bredesen. 1993. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 262:1274–1277. 29. Kastan, M. B., Q. Zhan, W. S. El-Deiry, F. Carrier, T. Jacks, W. V. Walsh, B. S. Plunkett, B. Vogelstein, and A. J. Forance, Jr. 1992. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxiatelangiectasia. Cell 71:587–597. 30. Klar, A. J. S., J. N. Strathern, and J. A. Abraham. 1984. Involvement of double-strand chromosomal breaks for mating-type switching in Saccharomyces cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 49:77–88. 31. Korsmeyer, S. 1992. Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80:879–886. 32. Kuerbitz, S. J., B. S. Plunkett, W. V. Walsh, and M. B. Kastan. 1992. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA 89:7491–7495. 33. Lane, D. P. 1992. p53, guardian of the genome. Nature (London) 358:15–16. 34. Lane, D. P. 1993. A death in the life of p53. Nature (London) 362:786–787. 35. Lee, S., B. Elenbaas, A. Levine, and J. Griffith. 1995. p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/ deletion mismatches. Cell 81:1013–1020. 36. Lee, S. E., C. R. Pulaski, D. M. He, D. M. Benjamin, M. J. Voss, J. Um, and E. A. Hendrickson. 1995. Isolation of mammalian cell mutants that are x-ray sensitive, impaired in DNA double-strand break repair and defective for V(D)J recombination. Mutat. Res. 336:279–291. 37. Lennon, S. V., S. J. Martin, and T. G. Cotter. 1991. Dose-dependent induc-
38. 39. 40. 41. 42. 43.
44. 45. 46. 47. 48.
49.
50. 51. 52. 53. 54. 55. 56. 57. 58.
59. 60. 61. 62. 63. 64. 65. 66.
5857
tion of apoptosis in human tumour cell lines by widely divergent stimuli. Cell Prolif. 24:203–214. Lowe, S. W., H. E. Ruley, T. Jacks, and D. E. Housman. 1993. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74:957–967. Lowe, S. W., E. M. Schmitt, S. W. Smith, B. A. Osborne, and T. Jacks. 1993. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature (London) 362:847–849. Murray, A. W. 1992. Creative blocks: cell-cycle checkpoints and feedback controls. Nature (London) 359:599–604. Oltvai, Z. N., and S. J. Korsmeyer. 1994. Checkpoints of dueling dimers foil death wishes. Cell 79:189–192. Oltvai, Z. N., C. L. Milliman, and S. J. Korsmeyer. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programed cell death. Cell 74:609–619. Pantazis, P., A. J. Kozielski, J. T. Mendoza, J. A. Early, H. R. Hinz, and B. C. Giovanella. 1993. Camptothecin derivatives induce regression of human ovarian carcinomas grown in nude mice and distinguish between non-tumorigenic and tumorigenic cells in vitro. Int. J. Cancer 53:863–871. Pietenpol, J. A., and B. Vogelstein. 1993. No room at the p53 inn. Nature (London) 365:17–18. Price, A. 1993. The repair of ionizing radiation-induced damage to DNA. Semin. Cancer Biol. 4:61–71. Radford, I. R. 1986. Evidence for a general relationship between the induced level of DNA double-strand breakage and cell-killing after X-irradiation of mammalian cells. Int. J. Radiat. Biol. 49:611–620. Raff, M. C. 1992. Social controls on cell survival and cell death. Nature (London) 356:397–400. Reynolds, J. E., T. Yang, L. Qian, J. D. Jenkinson, P. Zhou, A. Eastman, and R. W. Craig. 1994. Mcl-1, a member of the bcl-2 family, delays apoptosis induced by c-myc overexpression in Chinese hamster ovary cells. Cancer Res. 54:6348–6352. Sato, T., M. Hanada, S. Bodrug, S. Irie, N. Iwama, L. H. Boise, C. B. Thompson, E. Golemis, L. Fong, H.-G. Wang, and J. C. Reed. 1994. Interaction among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc. Natl. Acad. Sci. USA 91:9238–9242. Schwartz, D. C., and C. R. Cantor. 1984. Separation of yeast chromosomesized DNAs by pulse field gradient electrophoresis. Cell 37:67–75. Sen, S., and M. D’Incalci. 1992. Apoptosis. Biochemical events and relevance to cancer chemotherapy. FEBS Lett. 307:122–127. Sentman, C. L., J. R. Shutter, D. Hockenbery, O. Kanagawa, and S. J. Korsmeyer. 1991. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 67:879–888. Shimizu, S., Y. Eguchi, H. Kosaka, W. Kamiike, H. Matsuda, and Y. Tsujimoto. 1995. Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature (London) 374:811–813. Stamato, T. D., and N. Denko. 1990. Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells. Radiat. Res. 121:196–205. Strasser, A., A. W. Harris, and S. Cory. 1991. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell 67:889–899. Strasser, A., A. W. Harris, T. Jacks, and S. Cory. 1994. DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by bcl-2. Cell 79:329–339. Takayama, S., T. Sato, S. Krajewski, K. Kochel, S. Irie, J. A. Millan, and J. C. Reed. 1995. Cloning and functional analysis of BAG-1: a novel Bcl-2binding protein with anti-cell death activity. Cell 80:279–284. Taniguchi, S., Y. Hirabayashi, T. Inoue, M. Kanisawa, H. Sasaki, K. Komatsu, and K. J. Mori. 1993. Hemopoietic stem-cell compartment of the SCID mouse: double-exponential survival curve after g-irradiation. Proc. Natl. Acad. Sci. USA 90:4354–4358. Vaux, D. L., and I. L. Weissman. 1993. Neither macromolecular synthesis nor Myc is required for cell death via the mechanism that can be controlled by Bcl-2. Mol. Cell. Biol. 13:7000–7005. Vogelstein, B., and K. W. Kinzler. 1992. p53 function and dysfunction. Cell 70:523–526. Ward, J. F. 1988. DNA damage produced by ionizing radiation in mammalian cells: mechanisms of formation and reparability. Prog. Nucleic Acid Res. Mol. Biol. 35:95–125. Waring, P. 1990. DNA fragmentation induced in macrophages by gliotoxin does not require protein synthesis and is preceded by raised inositol triphosphate levels. J. Biol. Chem. 265:14476–14480. Weinert, T. A., and L. H. Hartwell. 1988. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science 241: 317–322. Wolf, D., and V. Rotter. 1985. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proc. Natl. Acad. Sci. USA 82:790–794. Wylie, A. H. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (London) 284:555–556. Yin, X.-M., Z. N. Oltvai, and S. J. Korsmeyer. 1994. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature (London) 369:321–323.
