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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 ICANÅ’RRBSIÃŒARCII53.4776^t7so,Octoberis.ITO] Advances in Brief 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.ïProgram, Division of Cancer Treatment ¡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 alíeleshave a much higher frequency of developing tumors than lines that showed minimal (., arrest, eight had mutant p53 alíeles,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 alíelessuch that each cell expresses one wild-type and following radiation. Studies with the hétérozygotesshowedthat the mu tant protein can have a dominant negative influence upon wild-type ¡>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 hétérozygoteswerealso 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 37°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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1993 American Association for Cancer Research. ROLE OF pS3 IN RADIOSENSITIVITY Table 1 Characteristics of the Burkitt's lymphoma and ¡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 lineWMNFWLNL2AG876SHOJLP119EW36AKUAST486CA46RamosSG568NamalwaP3HR1MCICell statusWTAVTWTAVTWTAVTWTAVTWTAVTWTAVTWTAVTWT/mutantWT/mutantMutantMutantMutantMutantMutantMutantMutantMutantExonmutation75775768786Alíelemutation248158248254175, (h)2425252130222421202123231817232326 typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeWild typeArg typeWild GinArgto typeWild HisArgto typeDeletedDeletedArg GinHeto AspRepeatedArgto 176248163287238234ChangeWild GinTyrto to TrpEBVNegativePositivePositivePositivePositiveNegativeNegativePositiveNegativeNegativeNegativeNegativePositivePositiveNegativePositiveNegativeDoubling HisGluto EndCysto 16HWLJD38CharacterizationBurkitt'sLymphoblastoidLymphoblastoidBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitl'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sBurkitt'sp53TyrTyrto to CysChangeWild " EBV, Epstein-Barr virus. 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 4°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 37°C,500 units/ml, Sigma Counter and channelyzer (Coulter Electronics, Hialeah, FL).
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