Role of the P53 Tumor Suppressor Gene in Cell Cycle Arrest and Radiosensitivity of Burkitt's Lymphoma Cell Lines

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Role of the p53 Tumor Suppressor Gene in Cell Cycle Arrest and Radiosensitivity of Burkitt's Lymphoma Cell Lines

Patrick M. O'Connor,1 Joany Jackman, Daniel Jondle, Kishor Bhatia, Ian Magrath, and Kurt W. Kohn

Laboratory of Molecular Pharmacology, Developmental Thcra[>eulic.Ã¯Program, Division of Cancer Treatment Â¡P.M. ()., J. J., D. J., K. W. A'./, and Lymphoiil Biology Section, Pediatrics Branch /K. B., I. M./, National Cancer Institute, N1H, Rethesda, Maryland 20892

Abstract ization. Several lines of evidence suggest that p53 functions by bind ing to DNA as an oligomer. Also, cells heterozygous for p53 form We have assessed the role of the p5ÃŒtumorsuppressor gene in cell cycle heterooligomeric complexes (mutant/wild-type p53 complexes), in arrest and cytotoxicity of ionizing radiation in 17 Burkitt's lymphoma and which the ability of the wild-type protein to function is suppressed Ivmphoblastoid cell lines. Cell cycle arrest was assessed by flow cytometry of cells 16 h following irradiation. In addition to the usual G2 arrest, the (11). It appears that the p53 protein is not required for normal mouse cell lines exhibited three types of responses in I.,: Class I, strong arrest in development since transgenic mice lacking both p53 genes are born G i following radiation; Class II, minimal arrest; and Class III, an inter normal (12). Nonetheless, knockout mice and mice expressing mutant mediate response. All Class I cells contained normal p53 genes. Of the ten p53 alÃeleshave a much higher frequency of developing tumors than lines that showed minimal (., arrest, eight had mutant p53 alÃeles,and two their wild-type counterparts (12, 13). These findings are reminiscent lines were heterozygous for p53 mutations. Both of the lines showing an of the predisposition of patients with Li-Fraumeni syndrome to mul intermediate response contained wild-type p53. Our results are consistent tiple neoplasms (14, 15). These patients suffer germ line mutations in with the view that mutations abrogate the ability ofp53 to induce (-, arrest one of the p53 alÃelessuch that each cell expresses one wild-type and following radiation. Studies with the hÃ©tÃ©rozygotesshowedthat the mu tant protein can have a dominant negative influence upon wild-type Â¡>5.1. one mutant p53 protein. Overexpression of wild-type p53 causes cells to arrest in G, of the and the reduced ability of two normal p53 lines to arrest in t., indicated that p53 function can be impaired by other mechanisms. The radiosensi- cell cycle, in accordance with inhibition by p53 of the initiation of DNA replication ( 16). It is now clear that wild-type p53 is required for tivity of most of the lines appeared to depend on the ability ofp53 to induce a G, arrest. The mean radiation dose that inhibited proliferation of the G, arrest following ionizing radiation; cells having mutant or no p53 Class I lines by 50% was 0.98 Gy. Of the eight pS3 mutant cell lines tested, genes fail to demonstrate this response (9, 17, 18). The above findings five lines required approximately 2.9 Gy to cause a 50% inhibition of cell suggest that p53 acts as a checkpoint control protein that halts the cell proliferation. The two hÃ©tÃ©rozygoteswerealso more resistant to radiation cycle in G, while DNA damage is present. This would presumably than the Class I cells (50% inhibitory dose, 2.1 and 2.9 Gy). Our results allow more time for DNA repair to be completed before progression suggest that radioresistance is afforded by a loss of function of wild-type into S phase. The role of p53 is in this sense analogous to that of the p53, which would normally induce a G| arrest and promote cell death in RAD9 gene, which in yeast inhibits progression of cells from G2 into the presence of DNA damage. mitosis following DNA damage (19). The participation of p53 and Introduction RAD9 in checkpoint controls that ensure fidelity in the transmission of genetic material from one cell generation to the next is supported by The p53 tumor suppressor gene is the most commonly mutated gene findings that cells lacking p53 or RAD9 activity exhibit a greater in human cancer (1-3). The normal gene product exerts antiprolifera- frequency of gene amplification/mutations than do wild-type cells (19, tive and antitransforming activity and in some cases promotes cell 20). The actions of p53 and RAD9 might also be expected to protect death via apoptosis. The precise mechanism by which p53 exerts its cells from the cytotoxic effects of DNA damaging agents. Abrogation actions is still unclear; however, p53 binds to specific DNA sequences of G2 arrest, either by genetic inactivation of RAD9 or with methyl- and can act both as a transcriptional activator and repressor (4-6). xanthines, increases the sensitivity of cells to DNA damaging agents Genes that wild-type p53 /raws-activates include the MDM2 gene, the (19, 21, 22), indicating that at least the G2 checkpoint plays a protec function of which appears to antagonize the activity of p53 (7), and the tive role against DNA damage induced cytotoxicity. GADD45 gene, which was originally identified by its coordinate In the present study we investigated whether activation of the p53 induction following growth arrest and DNA damage (8, 9). The spe dependent checkpoint in GÃŒwouldafford protection to ionizing ra cific DNA binding domain ofp53 resides within a central region of the diation. For this purpose we assayed 17 Burkitt's lymphoma and protein that contains putative metal binding sites that are important for lymphoblastoid cell lines for their ability to arrest in G| following maintenance of the wild-type p53 conformation (10). Mutations in 7-irradiation and correlated this response to the status of the p53 gene p53 cluster predominantly within this DNA binding region and lead to and radiosensitivity. The results suggest that contrary to expectation, a loss of function of both the DNA binding and biological activity of normal p53 function in Burkitt's lymphoma and lymphoblastoid cells the protein (1-5, 9-10). The half-life of wild-type p53 is on the order enhances radiosensitivity. A possible reason for this is discussed. of 20 min. However, mutant forms of p53 frequently have longer half-lives, leading to constitutively elevated levels of mutant p53 in Materials and Methods tumor cells (1-3). The COOH-terminal region of p53 contains nuclear localization sequences and a domain that is important for oligomer- Cell Culture. Burkitt's lymphoma and lymphoblastoid cell lines were de rived either at the National Cancer Institute from biopsies or normal periph eral lymphocytes, or from the American Type Culture Collection (Rockville, Rcccivcd 8/13/9.1; accepted 9/2/93. MD) or National Institute of General Medical Sciences cell repositories The costs of publication of this article were defrayed in part by the payment of page (Camden, NJ). Cells were grown at 37Â°Cin 95% air/5% CO2 in RPMI 1640 charges. This article must therefore he hereby marked advertisement in accordance with containing 15% heat-inactivated fetal bovine serum, 2 HIMt.-glutamine, 50 18 U.S.C. Section 1734 solely to indicate Ihis fact. 1To whom requests for reprints should be addressed, at Room 5C-25. Bldg. 37. units penicillin, and 50 fig/ml streptomycin. All tissue culture products were National Cancer Institute. Bcthesda. MD 20892. obtained from Advanced Biotechnologies (Columbia, MD) and routinely 4776

Table 1 Characteristics of the Burkitt's lymphoma and Â¡ymphoblastoid cell line panel Shown is the p53 status of each cell line as confirmed by single-strand conformation polymorphism analysis and DNA sequencing of exons 5 through 8 (23) and the status of each cell line with regard to the presence of the EBV genome.

time CelllineWMNFWLNL2AG876SHOJLP119EW36AKUAST486CA46RamosSG568NamalwaP3HR1MCIstatusWTAVTWTAVTWTAVTWTAVTWTAVTWTAVTWTAVTWT/mutantWT/mutantMutantMutantMutantMutantMutantMutantMutantMutantExonmutation75775768786AlÃelemutation248158248254175, (h)2425252130222421202123231817232326 typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild

typeWild typeWild typeArg typeWild

toGinArg typeWild toHisArg typeDeletedDeletedArg

toGinHe toAspRepeatedArg 176248163287238234ChangeWild toGinTyr to TrpEBVNegativePositivePositivePositivePositiveNegativeNegativePositiveNegativeNegativeNegativeNegativePositivePositiveNegativePositiveNegativeDoubling toHisGlu toEndCys 16HWLJD38CharacterizationBurkitt'sLymphoblastoidLymphoblastoidBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitl'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sp53toTyrTyr

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tion according to the manufacturer's recommendations (Amersham). Mono monitored for the presence of Mycoplasma contamination. The status of the p53 gene in the cell lines used was previously assessed by single-strand con clonal antibodies PAb 1801 and PAb 240 (Oncogene Science, Inc., Manhasset, formation polymorphism analysis of "hot spot" exons 5 through 8. This tech NY) recognize epitopes that reside between amino acids 32 and 79 and amino nique was used to identify the exon(s) harboring the mutations and then po- acids 212 and 217 of p53, respectively. lymerase chain reaction products of exons showing abnormal migration were Survival Studies. Cytotoxicity was determined from %-h growth inhibi subjected to direct sequencing (23). The results of these studies are shown tion assays as described previously (24). Briefly, exponentially growing cells in Table 1. (2 X laVml) were irradiated at room temperature (0.79-12.6 Gy) using a 137Cs Flow Cytometry. Cells were washed in ice-cold PBS,2 (pH 7.4, 5 ml), source delivering 5.25 Gy/min (1 Gy = 100 rads). Cells were postincubated fixed in 70% ethanol (5 ml), and stored at 4Â°C.Cells were then washed once and cell counts and cell size were determined every 24 h using a Coulter with ice-cold PBS (5 ml), treated with RNase (l h at 37Â°C,500 units/ml, Sigma Counter and channelyzer (Coulter Electronics, Hialeah, FL). Growth fraction Chemical Co., St. Louis, MO), and DNA was stained with propidium iodide was quantitated at the time the control untreated population had reached 8 (50 fig/ml). Cell cycle determination was performed using a Becton-Dickinson times the initial inoculum (3 cell doublings). Duplicate determinations were fluorescence-activated cell analyzer in which DNA content, as assayed by made within each of two independent experiments. propidium iodide staining, was used to distinguish each cell cycle phase. Quantitation was performed using the sum-of-broadened-rectangle model pro gram provided by the manufacturer: 3â€”5S-phase peaks were used to fit the model. DNA synthesis was also monitored at 16 h following irradiation by labeling the cells for 30 min with 10 /J.Mbromodeoxyuridine (Sigma). Follow ing bromodeoxyuridine labeling, cells were washed and fixed as described above. Cells were then resuspended in 1 ml 0.1 MHC1 containing 0.25% Triton X-100 at 4Â°Cand left on ice for 10 min. Cells were then diluted with 5 ml of distilled water and centrifuged and resuspended in 2 ml of water; DNA was denatured by boiling for 10 min. Afterwards cells were cooled on an ice slurry for 10 min, washed in 5 ml of PBS containing 0.25% Triton X-100, and then resuspended in 0.1 ml of PBS/Triton containing 5 ng/ml of an anti-bromodeoxyuridine-fluorescein conjugate (Boehringer Mannheim Biochemicals, India napolis, IN). Results were collected for a minimum of 15,000 cells for each determination. Gel Electrophoresis and Western Blotting. Cells were lysed on ice for 30 min in 1% Nonidet P-40 prepared in PBS that contained leupeptin (10 /j-g/ml), aprotinin (10 (Â¿g/ml),2 IHM4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mm sodium o-vanadate, 10 mM sodium fluoride, and 5 mm sodium pyrophosphate. Protein determination was performed using the BCA protein assay kit accord ing to the manufacturer's instructions (Pierce, Rockford, IL). Seventy-five fig of total cell protein were loaded onto sodium dodecyl sulfate-polyacrylamide mu un un gels and electrophoresed as described previously (22). Proteins were trans ferred to Immobilon membranes (Millipore, Bedford, MA) using semidry Fig. 1. Measurement of Gt arrest following y-irradiation in Burkitt's lymphoma and blotting techniques. Membranes were blocked for 30 min in 5% skim milk, lymphoblastoid cell lines. Shown is the percentage of the control GÂ¡population that re mained in G, for up to 16 h following 6.3 Gy radiation. Cell cycle distribution was probed for l h with primary antibodies, and then probed with sheep anti-mouse quantitated using flow cytometry as described in "Materials and Methods." Measure horseradish peroxidase second antibodies (Amersham Corporation, Arlington ments shown were made in the presence of nocodazole (0.4 ng/ml) to ensure that cells Heights, IL). Antibody reaction was revealed using chemiluminescence detec from the previous cell cycle would not reenter GÃ¬during the course of the experiment. Samples were grouped into three classes: Class I, strong arrest in GI following radiation (>60% of the original population); Class II, minimal arrest in G, following radiation 2 The abbreviations used are: PBS, phosphate buffered saline; ID5(I radiation dose (<25% of the original population); and Class III. an intermediate response. Abscissa, required to cause 50% inhibition of cell proliferation. status of the p53 gene. 4777

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0 -Wfflâ€”JiM 200 400 600 10' 102 103 400 600 10Â° 10' IO2 103 FL2-A FL1-H FL2-A FL1-H Fig. 2. Competence of G, arresi in Class I and lack of competence of G, arrest in Class II cells following 7-irradiation. Exponentially growing cells were irradiated with 6.3 Gy and then postincuhated for 16 h in the absence of nocodazole. A typical Class I response showing strong arrest in G, and G2 following irradiation is illustrated for the case of WMN cells. A typical Class II response showing minimal arrest in GÃŒ;nonetheless arrest in GT following irradiation is illustrated in the case of CA46 cells. Flow cytometric determination of cell cycle was performed hy staining the DNA with propidium iodide (PI, shown on the FL2 axis) and DNA synthesis was monitored using an fluorescein isothiocyanate labeled anti-hromodeoxyuridine antibody (FITC shown on the FLÃŒaxis).

Results and Discussion minimal G, arrest, eight had only mutant p53 alÃeles,and two lines were heterozygous, containing both mutant and normal p53 alÃeles. In the present study we assessed the role of Ihe p53 tumor suppres sor gene in cell cycle arrest and radiosensitivity of 17 Burkitt's lym- The remaining two cell lines (12%) showed an intermediate response: both of these lines by single-strand conformation polymorphism and phoma and lymphoblastoid cell lines. The purpose of these studies DNA sequencing contained only wild type p53 (23). Using this flow was to determine whether the presence of a normal p53 gene was cytometric approach we were able to predict correctly the status of the essential for cell cycle arrest in G, following DNA damage and p53 gene in 88% of the cases analyzed (15 correct of 17 tested). This whether the functional status of the p53 gene product would correlate approach therefore provides a simple means to assess the functional with the sensitivity of cells to ionizing radiation. status of p53 in cultured cell lines. The ability of cells to arrest in G, following radiation was assessed We found that the presence of Epstein-Barr virus in several of the by flow cytometry of cells 16 h following irradiation of exponentially wild type p53 lines tested did not significantly alter the ability of cells growing cultures with 6.3 Gy. This experiment was performed with to arrest in GI following irradiation (compare Fig. 1 with Table 1). coded samples so that the operators performing the analysis were This is contrary to the inhibition of p53 function observed in cells unaware of the p53 status of the cell lines until the results were infected with SV40 and human papilloma virus (1-3, 25). completed. Fig. 1 shows the results gathered from the 17 cell lines Our results are consistent with previous observations suggesting tested. As a matter of convenience, the cell lines were divided into that mutations in the p53 gene prevent cells from arresting in G] three classes on the basis of their G! arrest responses: Class I, strong following irradiation (17, 18). The behavior of the two lines that were arrest in G, following radiation (>60% of the original population); heterozygous for p53 suggests that the mutant protein has a dominant Class II, minimal arrest in G, following radiation (<25% of the negative influence upon the activity of the wild type p53 alÃele.In the original population); Class III, an intermediate response. Incubation of seven cases where p53 was determined to be wild type, two lines, cells with the mitotic inhibitor, nocodazole, following radiation (to JLP119 and EW36, showed a reduced ability to arrest in G! following trap any cells that might leak from G2 into G, of the next cell cycle) radiation. This suggested that JLP119 and EW36 cells have a defect in did not significantly alter the results, nor did increasing the radiation another part of the pathway that controls G, arrest following ionizing dose by a factor of 2. These findings confirmed that cells observed in radiation. Inactivation of p53 in these lines is not due to overexpres- G, at 16 h following radiation were arrested in G, and did not sion of MDM2 (7), since MDM2 mRNA levels differed less than represent cells leaking from G2 of the previous cell cycle. For illus 2-fold among the lines tested.3 In an attempt to discover the cause of trative purposes Fig. 2 has been included to show the contrasting G, this defect we analyzed the response of the p53 protein to ionizing responses of Class I and II cells following radiation. Both Class I and radiation. DNA damage induced by either ionizing radiation or UV II cells showed clearing of the S-phase population as evidenced by light leads to accumulation of the wild type p53 protein through a decreased bromodeoxyuridine staining and arrest in G2 phase of the mechanism that involves stabilization of the protein (17, 18, 26). Fig. cell cycle. 3A shows that exponentially growing cells expressing wild-type p53 When the coded samples were indexed according to their p53 status (Table 1), all of the five lines (29%) that showed strong G, arrest contained only normal p53 genes. Of the ten lines (59%) that showed ! K. Bhatia and I. Magrath, unpublished observations. 4778

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Fig. 3. Determination of p53 protein levels Â¡nBurkitt's lymphoma and lymphoblastoid cell lines. A, exponentially growing cells were lysed and 75 /xg of total cell protein from each cell line were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels, Western blotted, and probed with monoclonal antibodies raised against two independent epitope sites on the p53 protein (Oncogene Science). Antibody reaction was disclosed using the Amersham enhanced chemilumincscence detection system as described previously (22). B, effect of radiation on p53 protein levels. Exponentially growing cells were irradiated (6.3 Gy) and then incubaled for 4 h at 37Â°Cbefore cell lysis. Seventy-five (Â¿goftotal cell protein from each treatment was blotted and then probed with the monoclonal antibody PAblSOl (Oncogene Science). Along the top of ÃŸisthe p53 status of each cell line (data shown in Table 1) and the strength of G, arrest following radiation (data shown in Fig. 1). produce barely detectable levels of the protein in Western blotting. By range of 0.68-1.3 Gy (mean, 0.98 Gy; compare Figs. 1 and 4). Cell 4 h following radiation, however, the abundance of the p53 protein size determination of cultures throughout the assay revealed a dose increased markedly in three examples of Class I cells (Fig. 3fl). The and time dependent accumulation of cellular debris consistent with the two cell lines that by our sequencing criteria had normal p53 alÃeles cytotoxic action of the radiation exposure (results not shown). Of the but exhibited reduced ability to arrest in G, following radiation eight p53 mutant cell lines tested, five lines required 2.35-3.73 Gy of showed either no increase (JLP119) or only a small increase (EW36) radiation (mean, 2.89 Gy) to cause a 50% inhibition of cell prolifera in basal p53 protein levels following irradiation. The mechanism tion. The two lines that were heterozygous for p53 mutations also responsible for p53 stabilization is therefore defective in these lines. required higher radiation doses to kill an equivalent number of cells Analogous observations have been made in certain ataxia-telangiec- compared to the Class I cells (ID5II = 2.1 and 2.9 Gy for AKUA and tasia cell lines (9). We are presently endeavoring to characterize this ST486, respectively). The two cell lines that appeared to have normal defect in more detail. p53 alÃelesbut partiaily impaired G, delay, exhibited radiosensitivity Also shown in Fig. 3 is the response of four cell lines that are that was intermediate (JLP119 ID5() = 1.55 Gy, EW36 ID.,,, = 1.93 representative of Class II cells that exhibited minimal arrest in G, Gy) between Class I (mean, 0.98 Gy) and Class II (mean, 2.89 Gy) following irradiation (Fig. 1). Three of these cell lines (CA46, Ramos, cells. Thus, the majority of lines expressing either mutant or partially and MCI 16), like most mutant p53 lines we have tested, exhibited active forms of p53 were more resistant to radiation (approximately constitutively elevated levels of the mutant p53 protein compared to 2-3-fold) compared to their Class I counterparts. Our studies suggest the wild type lines (Fig. 3). These mutant p53 lines did not show any a positive correlation between the ability of cells to escape G, arrest further accumulation of the p53 protein following radiation. Some cell and radioresistance (compare Figs. 1 and 4). Three of the 17 cell lines lines (AKUA, HWL, and P3HR1) which had mutant p53 alÃelesdid tested did not comply with this relationship. MCI 16, HWL, and not exhibit constitutively elevated p53 protein levels. It therefore P3HR1 all expressed mutant p53 and failed to arrest in G, following seems that not all mutations in lymphoid tumors prolong the half-life radiation but were as radiosensitive as the Class I cells. We do not yet of the protein (23). When we tested the p53 response of the AKUA and know why these mutant p53 lines differed from the other Class II HWL lines to radiation we observed a significant accumulation of the cells. However, it is not unreasonable to suspect that factors other than mutant p53 protein (Fig. 3ÃŸ),indicating that the mechanism respon p53 will have a bearing on the outcome of radiation exposure. sible for stabilization of p53 is normal in these cells, although G, A potential explanation for the radioresistance observed in our arrest is defective. studies might be afforded by loss of function of the wild type p53 The cytotoxicity of ionizing radiation in the 17 lines was assessed protein, which would normally modulate the transcriptional activity of in 72-96-h growth inhibition assays as described in "Materials and genes to induce a G, arrest and/or promote cell death in the presence Methods." The radiation dose that inhibited proliferation of the Class of DNA damage. In agreement with this suggestion, the thymocytes of 1 wild type p53 cell lines by 50% of the control clustered within a transgenic mice having no p53 alÃelesare more resistant to apoptosis 4779

12: 2866-2871, 1992. â€¢¿3.75â€”-r. J Mack, D. H., Vartikar, J., Pipas, J. M., and Laimins, L. A. Specific repression of TATA-mediated but not initiator-mediated transcription by wild-type p53. Nature (Lond.), 363: 281-283, 1993. Xiangwei, Wu., Bayle, J. H., Olson, D., and Levine, A. J. The p53-mdm-2 auloregu- latory feedback loop. Genes & Dev., 7: 1126-1132, 1993. Fornace, A. J., Jr., Alamo, I., Jr., and Hollander, M. C. DNA damage-inducible .5.0^HE transcripts in mammalian cells. Proc. Nati. Acad. Sci. USA, 85: 8800-8804, 1988. Kastan, M. B., Zhan, Q., El-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V, Plunkett, B., Vogelstein, B., and Fornace, A. J., Jr. A mammalian cell cycle checkpoint E J038 utilizing p53 and GADD45 is defective in ataxia telangiectasia. Cell, 71: 587-597, (32.25_in2 NRMRlDJnOEI"Ã•6OJIPI'9 1992. Hainaut, P., and Milner, J. A structural role for metal ions in the "wild-type" confor mation of the tumor suppressor protein p53. Cancer Res., 53: 1739â€”1742,1993. 1.5.75ISGSSSâ€¢m,Qsi486IcfHb16A J MCI Milner, J., and Medcalf, E. A. Cotranslation of activated mutant p53 with wild type N12I drives the wild-type p53 protein into the mutant conformation. Cell, 65: 765-774, Â¡IHÂ»t'â„¢ 1991. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr., FINIIp53 lim B Butel, J. S., and Bradley, A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors. Nature (Lond.), 356: 215-221, 1992. Laviguer, A.. Maltby, V, Mock, D., Rossant, J., Pawson, T., and Bernstein, A. High incidence of lung, bone and lymphoid tumors in transgenic mice overexpressing MUTHNT6.7.8.9.10.11.12.13.GÃ¬un/un UJT/IDT UJT/MU mutant alÃelesof the p53 oncogene. Mol. Cell. Biol., 9: 3982-3991, 1989. flRREST +++ + - - 14 Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J. F. Jr., Nelson, C. E., Kim, D. H., Kassel, J., Gryka, M. A., Bischoff, F. Z., Tainsky, M. A., and Friend, S. H. Germ line Fig. 4. Relationship between p53 status and radiosensitivity of a panel of Burkitt's p53 mutations in a familial syndrome of breast cancer, sarcoma's and other neo lymphoma cell lines. Exponentially growing cells (2â€”3X lOs/ml) were irradiated (0.79- plasms. Science (Washington DC), 250: 1233-1238, 1990. 12.6 Gy) at a dose rate of 5.25 Gy/min at room temperature and then incubated for up to 15. Srivastava, S., Zou, Z., Pirollo, K., Blattner, W., and Chang, E. H. Germ-line trans 96 h at 37Â°C.Cell counts and cell sizes were recorded every 24 h following irradiation as mission of a cancer-prone family with Li-Fraumeni syndrome. Nature (Lond.), 348: described in "Materials and Methods." Values shown are the dose of radiation that caused 747-749, 1990. a 50% reduction in proliferation over the assay period. Each point represents the mean of 16. Michalovitz, D., Halevy, O., and Oren, M. Conditional inhibition of transformation two independent experiments in which duplicate measurements were made within each and of cell proliferation by a temperature-sensitive mutant ofp53. Cell, 62: 671â€”680, experiment. Along the x axis is shown the p53 status of each cell line (data shown in Table 1990. 1) and the strength of GÃŒarrestfollowing radiation (data shown in Fig. I). 17. Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W. Participation of p53 in the cellular response to DNA damage. Cancer Res., 57: 6304-6311, 1992. induced by radiation (27, 28). Wild type p53 has also been implicated 18. Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan, M. B. Wild-type p53 is a in triggering apoptosis in the presence of a constitutively active myc cell cycle checkpoint determinant following radiation. Proc. Nati. Acad. Sci. USA, gene (Ref. 29 and references therein). Since Burkitt's lymphomas S9: 7491-7495, 1992. 19. Weinert, T. A., and Hartwell, L. Characterization of RAD9 of Saccharomyces cer- constitutively express c-myc due to a chromosomal translocation (24, evisiae and evidence that its function acts post-translationally in cell cycle arrest after 30), the connection between wild type p53, c-myc, and apoptosis DNA damage. Mol. Cell. Biol., 10: 6554-6564, 1990. 20. Livingstone, L. R., White, A., Sprouse, J., Livanos, E., Jacks, T., and Tlsty, T. D. might be broken by loss of function mutations in the p53 gene. This Altered cell cycle arrest and gene amplification potential accompany loss of wild-type might in turn render cells more resistant to radiation. We are presently p53. Cell, 70: 923-935, 1992. exploring the relationship between p53 and apoptosis in this system. 21. Fingert, H. J., Chang, J. D., and Pardee, A. B. Cytotoxic, cell cycle and chromosomal effects of methylxanthines in human tumor cells treated with alkylating agents. In summary, our findings suggest that the ability of p53 to induce Cancer Res., 46: 2463-2467, 1986. G] arrest following ionizing radiation correlates, in most cases, with 22. O'Connor, P. M., Ferris, D. K., Pagano, M., Draetta, G., Pines, J., Hunter, T., Longo, the radiosensitivity of Burkitt's lymphoma or lymphoblastoid cell D. L. and Kohn, K. W. G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin Bl/cdc2-kinase complexes differently. J. Biol. Chcm., 265: lines. The p53 protein in these cells, therefore, operates in a role that 8298-8308, 1993. is contrary to the actions of RAD9 at the G2 checkpoint. Activation of 23. Bhatia, K., Goldschmidts, W., Gutierrez, M., Gaidano, G., Dalla-Favera, R., and Magrath, I. Hemi- or homozygosity: a requirement for some but not other p53 mutant the G2 checkpoint tends to protect cells from the cytotoxicity of DNA proteins to accumulate and exert a pathogenic event. FASEB J., 7: 951-956, 1993. damaging agents (19, 21), while activation of the G, checkpoint 24. O'Connor, P. M., Wassermann, K., Sarang, M., Magrath, I., Bohr, V. A., and Kohn, K. appears to increase the cytotoxicity of ionizing radiation, at least in W. Relationship between DNA crosslinks, cell cycle and apoptosis in Burkitt's lym cells that have deregulated c-myc. Dissection of the downstream tar phoma cell lines differing in sensitivity to nitrogen mustard. Cancer Res., 51: 6550- 6557, 1991. gets of p53 action in G, arrest and apoptosis may provide useful 25. Kessis, T., Slebos, R. J., Nelson, W. G., Kastan, M. B., Plunkett, B. S., Han, S. M., information for the development of new chemotherapeutic approaches Lorincz, A. T., Hedrick, L., and Cho, K. R. Human papillomavirus 16 E6 expression disrupts the p53 mediated cellular response to DNA damage. Proc. Nati. Acad. Sci. to kill cancer cells selectively. USA, 90: 3988-3992, 1993. 26. Maltzman, W., and Czyzyk, L. UV irradiation stimulates levels of p53 cellular antigen References in nontransformed mouse cells. Mol. Cell. Biol., 4: 1689-1694, 1984. 27. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. p53 is 1. Hollstein, M.. Sidransky. D., Vogelstein. B., and Harris, C. C. p53 mutations in human required for radiation-induced apoptosis in mouse thymocytes. Nature (Lond.), 362: cancers. Science (Washington DC), 253.' 49-52, 1991. 847-849, 1993. 2. Vogelstein, B., and Kinzler, K. W. p53 function and dysfunction. Cell, 70: 523-526, 28. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. 1992. L., and Wyllie, A. H. Thymocyte apoplosis induced by p53-dependent and indepen 3. Levine, A., Momand, J.. and Finlay, C. A. The p53 tumor suppressor gene. Nature dent pathways. Nature (Lond.), 362: 849-852, 1993. (Lond.), 351: 453-456, 1991. 29. Wang, Y., Ramqvist, T., Szekely, L., Axelson, H., Klein, G., and Wiman, K. G. 4. El-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., and Vogelstein, B. Reconstitution of wild-type p53 expression triggers apoptosis in a p53-negative v-myc Definition of a consensus binding site forp53. Nat. Gen., l: 45â€”49,1992. retrovirus-induced T-cell lymphoma line. Cell Growth & Differ., 4: 467^73, 1993. 5. Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., and Shay, J. W. A transcrip- 30. Magrath, I. The pathogenesis of Burkitt's lymphoma. Adv. Cancer Res., 5: 133-270, tionally active DNA-binding site for human p53 protein complexes. Mol. Cell. Biol., 1990.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1993 American Association for Cancer Research. Role of the p53 Tumor Suppressor Gene in Cell Cycle Arrest and Radiosensitivity of Burkitt's Lymphoma Cell Lines

Patrick M. O'Connor, Joany Jackman, Daniel Jondle, et al.

Cancer Res 1993;53:4776-4780.

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