ARTICLE |
Correspondence to: Chin-Tarng Lin, Inst. of Pathology, College of Medicine, National Taiwan University, Taipei 10000, Taiwan, R. O. C.
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Summary |
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Recently, we have established nine nasopharyngeal carcinoma (NPC) cell lines in which only one cell line showed the p53 mutation. For investigation of the p53 mutation in this line, immunostaining using anti-p53 antibody was applied and showed the presence of p53 protein in the cytoplasm but not in the nucleus. Single strand conformation polymorphism analysis of the p53 gene showed one normal and one additional DNA band. Cloning and sequencing of PCR-amplified DNA showed an AGA (arginine) to ACA (threonine) heterozygous point mutation at codon 280. Transfection of the p53 DNA binding sequence and chloramphenicol acetyltransferase assay revealed loss of transcriptional activation function of endogenous p53 protein. Co-localization of the endogenous and the transfected exogenous p53 protein by polyclonal antibodies to anti-p53 protein revealed strong exogenous p53 staining in the transfected nuclei and weak staining of endogenous p53 protein in the cytoplasm. We concluded that (a) a heterozygous point mutation at codon 280 was identified in the NPC-TW 06 cell line; (b) the point mutation may cause the stagnation of mutant p53 protein in the cytoplasm, and loss of its transcriptional activation function; (c) endogenous and exogenous p53 protein can be co-localized at the same time in the transfected cells; and (d) 280 mutant p53 protein in NPC cells does not cause a decrease or increase in sensitivity to chemotherapy. (J Histochem Cytochem 45:991-1003, 1997)
Key Words: p53 point mutation, nasopharyngeal carcinoma cell line, immunohistochemical localization of transfected p53 protein, single strand conformation polymorphism, transfection of constructed plasmids, chloramphenicol acetyltransferase assay, cisplatin treatment
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Introduction |
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Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in Taiwan, South China, Hong Kong, and Singapore (
The p53 gene encodes a nuclear protein composed of 393 amino acids, is located on chromosome 17p (region 17p13), and contains 11 exons (the first exon does not encode protein sequence) (
Most NPC biopsy specimens show a normal p53 gene (
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Materials and Methods |
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Cell Cultures
The NPC cell line designated NPC-TW 06 was established and characterized in our laboratory previously (
Immunohistochemical Study
The NPC, U-2OS, and colon adenocarcinoma cell lines were immunostained using a previously described method (
Southern and Northern Blot Analyses
The isolation of genomic DNA from NPC culture cells was performed as previously described (
Synthesis of DNA Primers and Oligonucleotides
Oligonucleotide primers were synthesized by the phosphoramidites method using Model 380 Applied Biosystems DNA Synthesizers from the Institute of Biomedical Sciences (IBMS), Academia Sinica, Taipei. After deprotection by ammonia water, the oligonucleotides were purified by affinity column kits. Some primers were purchased from TIB MOLBIL (Berlin, Germany). The oligonucleotide sequence for PCR primers in exons 1-11 and sequencing primers in exons 8-9 were used according to published materials (
PCR Analysis
Genomic DNA (500 ng) was amplified by PCR using both p53-specific primers (
SSCP Analysis
The procedure for SSCP was performed according to a published protocol (
Clone Sequencing
Genomic DNA (0.5 µg) prepared from the NPC cell line was used in 100 µl PCR reaction mixture. The DNA cloning sequences at exon 8-9 for PCR primers were 5'-GGGAAGCTTTTGGGAGTAGATGGAGCCT-3' and 5'-GGGGAATTCAGTGTTAGACTGGAAACTT-3'. The PCR products were digested with EcoRI and HindIII, fractionated by 2% agarose gel, and specific bands electroeluted. The purified products were ligated into an EcoRI-HindIII-cleaved pGEM3 vector (Promega; Madison, WI). After bacterial transformation, at least four different p53 exon 8-9 subclones from the cell line were selected for sequencing. For comparison, normal control DNA from human peripheral blood cells was also included.
Two approaches were used to determine the DNA sequences of selected clones. The first approach was by direct sequencing of the amplified DNA via Sanger's dideoxy method using one of the sequencing primers (
The DNA sequences obtained were then compared with published p53 sequences for sequence homology using the sequence analysis software package of the Genetics Computer Group (Version 6.0, 1989; University of Wisconsin Biotechnology Center, Madison, WI).
Construction of Reporter Plasmid 3PBS-CAT
The palindromic oligonucleotide of p53 consensus DNA binding sequence (5'-AGCTAGGCATGTCTAGACATGCCT-3') (
Construction of Plasmids Containing Wild and Mutant p53 Genes
The wild-type and mutant p53 gene cloning from our NPC cell line was performed using methods similar to those mentioned above. The primers for cloning of NPC-TW 06 p53 wild-type and mutant type genes were 5'-GGGAAGCTTGCCGCCATGCATCATCATCATCATCATATGGAGGAG-CCGCAGT-3' and 5'-GGGGGATCCTCAGTCTGAGTCAGGCCC-3', which included the whole coding sequence (1.3 KB). The PCR products of cDNA were inserted into the pCEP4 vector, which contained CMV IE gene as the promoter.
Transfection
Using the calcium phosphate procedure (
CAT Assay
Cells transfected with different plasmids were harvested, washed twice in PBS, and resuspended in 100 µl of 0.5% Triton X-100, 0.25 M Tris-HCl (pH 8.0) at 4C, for 2 min. After the cells were spun for 5 min in an Eppendorf microfuge at 4C, the supernatants were removed and assayed for enzyme activity (
Assay for Cell Viability in wt-p53 and 280 mt-p53 Transfected NPC Cells After Cisplatin Treatment
Cell viability was assessed by MTT [3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma] assay (
cell viability = (experimental value - X)/(control value - X)
where X was the value obtained in wells containing medium and MTT without cells.
The relative survival rate of wt-p53 transfectant to survival rate of mt-p53 transfectant after cisplatin treatment was obtained compared with the survival rate of non-transfected NPC-TW 06 cells after cisplatin treatment.
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Results |
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Immunolocalization of p53 in the NPC Cell Line and Its Original Biopsy Specimen
When we used an MAb against mutant p53 (PAb240) to stain our NPC cell line, reaction product of anti-p53 was seen in the cytoplasm but not in the nucleus of each single cell (Figure 1A). Some cells revealed strong cytoplasmic staining, whereas others showed rather weak staining. All mitotic cells showed strong cytoplasmic but not chromosomal staining (data not shown). NPC cells starved in culture medium containing 0.2% FCS for 2 days showed nuclear but not cytoplasmic staining (Figure 1B). When the original culture medium containing fetal calf serum was used to reincubate with these starved cells for another 2 days, the p53 protein was localized in the cytoplasm (data not shown). When the cells were treated with cycloheximide, the p53 protein was localized to the nucleus (Figure 1C). If NPC cells were treated with cisplatin (5 x 10-5 M) for 1 day, the p53 protein was still localized in the cytoplasm (Figure 1D). When the original biopsy specimen from which the cell line was established was stained by the same antibody, the paraffin section (NPC-TW 06) revealed the same pattern of cytoplasmic but not nuclear staining in some tumor cells (Figure 2). Other tumor cells in the same specimen were not stained. When the positive control cells (colon adenocarcinoma) were stained with PAb240, both cytoplasmic and nuclear staining could be identified. If nonspecific ascites was used to stain the NPC cell line and colon cancer cell line, none of the tumor cells was specifically stained (data not shown).
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Southern and Northern Blot Analyses of p53 DNAs and RNAs in the NPC Cell Line
When the NPC high molecular weight DNA was digested with PvuII and analyzed by a p53 DNA probe, three bands of 10.5, 3.9, and 1.6 KB were identified in the NPC cell line and in the positive control DNA from normal human peripheral blood cells. A negative control cell line (hepatoma Hep3B) revealed only two bands, of 10.5 and 1.6 kb (data not shown). In Northern blot analysis, the NPC cell line showed a p53 band, whereas the negative control hepatoma Hep3B cell line revealed no p53 band (data not shown).
PCR and SSCP Analyses of the p53 Gene in the NPC Cells
In the gel analysis of PCR-amplified products from 11 exons, no specific abnormal band could be identified compared with p53 products from normal human peripheral blood cells (NHB) (data not shown). However, by SSCP, two distinct bands, one moving faster (F band) and the other more slowly (S band), could be clearly identified. The density of the F band was stronger than that of the S band. The mobility of the S band in the NPC line was similar to that of the normal control DNA (NHB cells) (Figure 3).
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Cloning and Sequencing
After the pGEM3 vectors, which had been inserted with our PCR products from the exon 8-9 region, had been transfected into the E. coli MC1061, four clones of bacteria from the culture plates were selected and amplified. The amplified DNA fragments were sequenced by both direct and automatic sequencing. Direct sequencing from some clones showed a point mutation at codon 280 from AGA to ACA (Arg&Aelig;Thr) (Figure 4B), whereas some other clones revealed normal sequence (Figure 4C), similar to the normal control DNA sequence (Figure 4A). The automatic sequencing showed the same results.
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CAT Assay
The p53 consensus binding sequence was cloned into the CAT plasmid, then transfected into the NPC cells, U-2OS cells, and Saos-2 cells. The CAT activity from each transfected cell line was then examined by thin-layer chromatography. Results showed that the NPC cell line (Figure 5, Lane 3) and the Saos-2 cell line (Figure 5, Lane 2) contained no CAT activity, whereas the U-2OS cell line (Figure 5, Lanes 1 and 5) had clear CAT activity. When both 3PBS-CAT plasmids plus pCEP4-wt p53 (pCEP4-wild type p53) or plus pCEP4-mt 280p53 (pCEP4-mutant type p53) were co-transfected into Saos-2 cells, CAT activity was seen only in the cells transfected with wt p53 plasmid (Figure 6A, Lane 1), and not in the cells transfected with mutant 280 p53 plasmid (Figure 6A, Lane 2). When 3PBS-CAT plasmid and wild-type p53-pCEP4 plasmid were co-transfected to the NPC cells, rather weak CAT activity was present (Figure 6B, Lane 1) compared with that in 3PBS-CAT-transfected U-2OS cells (Figure 6B, Lane 4). However, when the mt 280 p53-pCEP4 plasmid was used to replace wild-type p53-pCEP4 plasmid, no CAT activity could be seen in the NPC cells (Figure 6B, Lane 2). A summary of CAT assay results is shown in Table 1. The transfection efficiency of each constructed plasmid to the cells was judged by the immunostaining of p53 protein in the transfected cells (see below).
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Identification of Wild-type (wt) and Mutant (mt) 280 p53 Protein in Saos-2 and NPC Cells
When the Saos-2 cells were transfected with a wild-type p53 gene and the expression of normal p53 protein in the cells was detected by immunohistochemical localization, reaction product of anti-wt-p53 protein was present mostly in the nucleus and very weakly in the cytoplasm. The cells containing strong nuclear staining always showed a rounded appearance. When the cells were cultured for several days, those rounded cells revealed degenerative change. When the mt 280 p53 gene was transfected, the distribution of mt 280-p53 protein in the Saos-2 cells was the same as that of the wt-p53 protein. Even when a one-to-one ratio of wt-p53 and mt 280-p53 genes was transfected, the distribution of p53 was similar to that of individual transfections of wt-p53 or mt 280-p53 gene (data not shown). When NPC cells were transfected with the wt-p53 gene, the p53 protein could be observed both in the nuclei and cytoplasm of those cells containing the exogenous wt-p53 gene, with some cells containing strong nuclear and cytoplasmic staining and other cells with strong nuclear and weak cytoplasmic staining. The cells containing strong nuclear and cytoplasmic staining also showed a rounded morphology. When they were cultured for more than 3 days, most of them exhibited degenerative change. In contrast, the untransfected cells displayed only weak cytoplasmic staining (Figure 7A, small arrows). When the mt 280-p53 gene was transfected, a similar staining pattern was seen in the wt-p53 gene-transfected NPC cells (Figure 7B), except that the degenerative cells were fewer than those of the wt-p53 gene transfection). A summary of p53 protein distribution in the Saos-2 and NPC cells is given in Table 2. The number of strongly nuclear-stained tumor cells was about 40-60% of the total cell population in each experiment, indicating that the transfection efficiency in our experiment was about 50%.
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Assay for Cell Viability in wt-p53 and 280 mt-p53-transfected NPC Cells after Cisplatin Treatment
The IC50 of cisplatin to NPC cells was 5 x 10-5 M. When wt-p53 transfectants and nontransfected NPC cells were treated with IC50 of cisplatin for 24 hr, a ratio of 0.66 was identified by the MTT assay. Similarly, if the 280 mt-p53 transfectants were used to replace the wt-p53 transfectants, a ratio of 0.70 was obtained.
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Discussion |
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Immunolocalization of p53 protein in many tumors has been reported previously (
The abnormality of p53 protein in NPC cell line does not necessarily mean that it is also abnormal in a patient's tumor tissue. To clarify this situation, we immunostained sections of the paraffin block from which our NPC cell line had been established. The sections revealed similar cytoplasmic but not nuclear staining in certain tumor cells, but other tumor cells in the same section showed no p53 reaction product. These findings suggest that the NPC-TW 06 patient's tumor tissue may contain two types of tumor cells: some cells with a mutated p53 gene and other cells with a wild-type p53 gene. They also suggest that the mutation of the p53 gene in these NPC cells does not occur in the initiation stage of tumorigenesis.
Our results from Southern blotting to investigate whether the p53 gene was abnormal suggest that no specific gross rearrangement or deletion of the p53 gene exists in this NPC cell line. Similarly, Northern blot analysis also indicates that transcription of the p53 gene is unproblematic, unlike that in the osteogenic sarcoma (Saos-2) and hepatoma (Hep3B) cell lines, which showed notable p53 deletion (
The wild-type p53 protein can form a tetramer with a mutant p53 protein (
To be sure that most wild-type p53 protein molecules have been trapped in the cytoplasm, resulting in loss of p53 protein in the nucleus to perform its physiological function, we transfected a CAT plasmid construct containing a p53 DNA binding sequence into our NPC and Saos-2 (with deletion of p53 gene) cells. No CAT activity was detected in our NPC and Saos-2 cell lines, in contrast to the positive control cell lines U-2OS (with normal p53 gene), in which the CAT activity was easily detected. This result strongly indicates that, in NPC cells, neither wild-type p53 protein nor mutant p53 protein could get into the nucleus to activate its target genes. To determine whether the mutant p53 gene product in the NPC cell line had lost its transcriptional activation function, we co-transfected mutant p53-pCEP4 plasmid and 3PBS-CAT reporter plasmid into the NPC cells, which resulted in no CAT activity. However, NPC cells that had been transfected with mutant 280 p53 gene showed strong exogenous mutant p53 protein in their nuclei, suggesting that although the excess exogenous mutant p53 protein can not totally be bound by the limited cytoplasmic factor(s) and can move into the nuclei, this protein still can not activate the CAT gene to synthesize enzyme. Similarly, co-transfection of mutant p53-pCEP4 plasmid and 3PBS-CAT reporter plasmid into the Saos-2 cells also showed no activity of CAT enzyme, whereas the exogenous mutant p53 protein could also be detected in the nuclei of transfected Saos-2 cells. Both experiments strongly indicate that our NPC mutated p53 protein has already lost its transcriptional activation function.
To evaluate NPC cellular factors that may play a role in trapping the mutant and wild-type p53 protein complex, we also co-transfected wild-type p53-pCEP4 and 3PBS-CAT plasmids into both Saos-2 and NPC cells. Our results, showing that co-transfection of wild-type p53 and 3PBS-CAT into the p53-deleted Saos-2 cells exhibited strong CAT activity, whereas co-transfection into the NPC cells revealed weak CAT activity, suggest that some wild-type p53 protein may also be trapped in the NPC cytoplasm and that only part of its excess protein which is not bound by the saturated endogenous mutant 280 p53 protein can get into the nucleus to activate the CAT gene. This interpretation is supported by the results from immunostaining of the transfected cells, which showed strong exogenous mutant p53 protein in both the nuclei and cytoplasm in some cells and strong staining in the nuclei and weak in perinuclear region in other cells (Figure 7A). The findings that most wild-type p53 gene-transfected Saos-2 and NPC cells showed fewer cytoplasmic processes with smaller cell bodies (rounded appearance) and faster degeneration indicate that excess wild-type p53 protein may cause apoptotic change, a finding similar to that of a previous report (
The mechanism(s) of cytoplasmic accumulation of p53 protein in NPC cells is not yet well defined (
Although cancer cells treated with cisplatin may induce nuclear accumulation of wt p53 (
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Acknowledgments |
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Supported in part by a research grant (NSC85-2331-B-002-188-M06) from the National Science Council (CTL), by one from Academia Sinica, Taipei (CTL), and another from National Taiwan University Hospital (INTPRO-08) (JKH), Taipei, Taiwan, R. O. C.
We are grateful to Drs Y.S. Lin and T. Tan of the Institute of Biomedical Sciences, Academia Sinica, Taipei, and to Dr J.Y. Shew of Department of Biochemistry, College of Medicine, National Taiwan University, for helpful criticism. We also thank Ms H.M. Hwang for technical assistance.
Received for publication August 12, 1996; accepted January 12, 1997.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersson-Anvret M, Forsby N, Klein G, Henle W (1977) Relationship between the Epstein-Barr virus and undifferentiated nasopharyngeal carcinoma: correlated nucleic acid hybridization and histopathological examination. Int J Cancer 20:486-494[Medline]
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1988) Current Protocols in Molecular Biology. New York, Chichester, Brisbane, Toronto, Singapore, Greene Publishing Associates and Wiley Interscience
Baker SJ, Markowitz S, Fearon ER, Willson JK, Vogelstein B (1990) Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249:912-915[Medline]
Bartek J, Bartkova J, Lukas J, Staskova Z, Vojtesek B, Lane DP (1993) Immunohistochemical analysis of the p53 oncoprotein on paraffin sections using a series of novel monoclonal antibodies. J Pathol 169:27-34[Medline]
Birnboim HC (1988) Rapid extraction of high molecular weight RNA from cultured cells and granulocytes for Northern analysis. Nucleic Acids Res 16:1487-1497[Abstract]
Bosari S, Viale G, Roncalli M, Graziani D, Borsani G, Lee AK, Coggi G (1995) p53 gene mutations, p53 protein accumulation and compartmentalization in colorectal adenocarcinoma. Am J Pathol 147:790-798[Abstract]
Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M (1990) Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc Natl Acad Sci USA 87:973-1977[Abstract]
Brooks L, Yao QY, Rickinson AB, Young LS (1992) Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 66:2689-2697[Abstract]
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47:936-942[Abstract]
Caron de Fromentel C, Soussi T (1992) TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosomes Cancer 4:1-15[Medline]
Casey G, Lo-Hsueh M, Lopez ME, Vogelstein B, Stanbridge EJ (1991) Growth suppression of human breast cancer cells by the introduction of a wild-type p53 gene. Oncogene 6:1791-1797[Medline]
Chandar N, Billig B, McMaster J, Novak J (1992) Inactivation of p53 gene in human and murine osteosarcoma cells. Br J Cancer 65:208-214[Medline]
Chang YS, Tyan YS, Liu ST, Tsai MS, Pao CC (1990) Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification. J Clin Microbiol 28:2398-2402[Medline]
Chen PL, Chen YM, Bookstein R, Lee WH (1990) Genetic mechanisms of tumor suppression by the human p53 gene. Science 250:1576-1580[Medline]
Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC, Hooper ML, Wyllie AH (1993) Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362:849-852[Medline]
De The G (1982) The Herpes Viruses. Vol 1A. New York, Plenum
Di Nocera PP, Dawid IB (1983) Transient expression of genes introduced into cultured cells of Drosophila. Proc Natl Acad Sci USA 80:7095-7098[Abstract]
Diller L, Kassel J, Nelson CE, Gryka MA, Litwak G, Gebhardt M, Bressac B, Ozturk M, Baker SJ, Vogelstein B, Friend SH (1990) p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol 10:5772-5781[Medline]
Effert P, McCoy R, Abdel-Hamid M, Flynn K, Zhang Q, Busson P, Tursz T, Liu E, Raab-Traub N (1992) Alterations of the p53 gene in nasopharyngeal carcinoma. J Virol 66:3768-3775[Abstract]
El-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B (1993) Definition of a consensus binding site for p53. Nature Genet 1:45-49[Medline]
Eliyahu D, Michalovitz D, Eliyahu S, Pinhasi-Kimhi O, Oren M (1989) Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci USA 86:8763-8767[Abstract]
Fields S, Jang SK (1990) Presence of a potent transcription activating sequence in the p53 protein. Science 249:1046-1049[Medline]
Finlay CA, Hinds PW, Levine AJ (1989) The p53 proto-oncogene can act as a suppressor of transformation. Cell 57:1083-1093[Medline]
Finlay CA, Hinds PW, Tan TH, Eliyahu D, Oren M, Levine AJ (1988) Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life. Mol Cell Biol 8:531-539[Medline]
Friedman PN, Chen X, Bargonetti J, Prives C (1993) The p53 protein is an unusually shaped tetramer that binds directly to DNA. Proc Natl Acad Sci USA 90:3319-3323[Abstract]
Fritsche M, Haessler C, Brandner G (1993) Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene 8:307-318[Medline]
Gannon JV, Greaves R, Iggo R, Lane DP (1990) Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J 9:1595-1602[Abstract]
Gannon JV, Lane DP (1991) Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport. Nature 349:802-806[Medline]
Gorman CM, Moffat LF, Howard BH (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044-1051[Medline]
Hinds PW, Finlay CA, Quartin RS, Baker SJ, Fearon ER, Vogelstein B, Levine AJ (1990) Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes. Cell Growth Differ 1:571-580[Abstract]
Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253:49-53[Medline]
Hsu YS, Tang FM, Liu WL, Chuang JY, Lai MY, Lin YS (1995) Transcriptional regulation by p53. Functional interactions among multiple regulatory domains. J Biol Chem 270:6966-6974
International Agency for Research on Cancer and International Association of Cancer Registries (1976) Cancer Incidence in Five Continents. Geneva, World Health Organization
Isaacs WB, Carter BS, Ewing CM (1991) Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. Cancer Res 51:4716-4720[Abstract]
Johnson P, Gray D, Mowat M, Benchimol S (1991) Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells. Mol Cell Biol 11:1-11[Medline]
Kern SE, Pietenpol JA, Thiagalingam S, Seymour A, Kinzler KW, Vogelstein B (1992) Oncogenic forms of p53 inhibit p53-regulated gene expression. Science 256:827-830[Medline]
Klein G, Giovanella BC, Lindahl T, Fialkow PJ, Singh S, Stehlin JS (1974) Direct evidence for the presence of Epstein-Barr virus DNA and nuclear antigen in malignant epithelial cells from patients with poorly differentiated carcinoma of the nasopharynx. Proc Natl Acad Sci USA 71:4737-4741[Abstract]
Lehman TA, Bennett WP, Metcalf RA, Welsh JA, Ecker J, Modali RV, Ullrich S, Romano JW, Appella E, Testa JR (1991) p53 mutations, ras mutations, and p53-heat shock 70 protein complexes in human lung carcinoma cell lines. Cancer Res 51:4090-4096[Abstract]
Levine AJ, Momand J, Finlay CA (1991) The p53 tumour suppressor gene. Nature 351:453-456[Medline]
Lin CT, Chan WY, Chen W, Huang HM, Wu HC, Hsu MM, Chuang SM, Wang CC (1993) Characterization of seven newly established nasopharyngeal carcinoma cell lines. Lab Invest 68:716-727[Medline]
Lin CT, Chan WY, Chen W, Shew JY (1992) Nasopharyngeal carcinoma and retinoblastoma gene expression. Lab Invest 67:56-70[Medline]
Lin CT, Dee AN, Chen W, Chan WY, Hsu MM (1994) The association of Epstein-Barr virus, human papilloma virus and cytomegalovirus in nasopharyngeal carcinoma cell lines. Lab Invest 71:731-736[Medline]
Lin CT, Wong CI, Chan WY, Tzung KW, Ho JK, Hsu MM, Chuang SM (1990) Establishment and characterization of two nasopharyngeal carcinoma cell lines. Lab Invest 62:713-724[Medline]
Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE, Jacks T (1994) p53 status and the efficacy of cancer therapy in vivo. Science 266:807-810[Medline]
Mayall FG, Goddard H, Gibbs AR (1992) p53 immunostaining in the distinction between benign and malignant mesothelial proliferations using formalin-fixed paraffin sections. J Pathol 168:377-381[Medline]
Mercer WE, Amin M, Sauve GJ, Appella E, Ullrich SJ, Romano JW (1990a) Wild type human p53 is antiproliferative in SV40-transformed hamster cells. Oncogene 5:973-980[Medline]
Mercer WE, Shields MT, Amin M, Sauve GJ, Appella E, Romano JW, Ullrich SJ (1990b) Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53. Proc Natl Acad Sci USA 87:6166-6170[Abstract]
Moll UM, LaQuaglia M, Benard J, Riou G (1995) Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc Natl Acad Sci USA 92:4407-4411[Abstract]
Moll UM, Riou G, Levine AJ (1992) Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc Natl Acad Sci USA 89:7262-7266[Abstract]
Nonoyama M, Huang CH, Pagano JS, Klein G, Singh S (1973) DNA of Epstein-Barr virus detected in tissue of Burkitt's lymphoma and nasopharyngeal carcinoma. Proc Natl Acad Sci USA 70:3265-3268[Abstract]
Porter PL, Gown AM, Kramp SG, Coltrera MD (1992) Widespread p53 overexpression in human malignant tumors. An immunohistochemical study using methacarn-fixed, embedded tissue. Am J Pathol 140:145-153[Abstract]
Raab-Traub N, Flynn K, Pearson G, Huang A, Levine P, Lanier A, Pagano J (1987) The differentiated form of nasopharyngeal carcinoma contains Epstein-Barr virus DNA. Int J Cancer 39:25-29[Medline]
Raab-Traub N, Hood R, Yang CS, Henry B, Pagano JS (1983) Epstein-Barr virus transcription in nasopharyngeal carcinoma. J Virol 48:580-590[Medline]
Raycroft L, Wu HY, Lozano G (1990) Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science 249:1049-1051[Medline]
Rotter V, Prokocimer M (1991) p53 and human malignancies. Adv Cancer Res 57:257-272[Medline]
Spruck CH, Tsai YC, Huang DP, Yang AS, Rideout WM, Gonzalez-Zulueta M, Choi P, Lo KW, Yu MC, Jones PA (1992) Absence of p53 gene mutations in primary nasopharyngeal carcinomas. Cancer Res 52:4787-4790[Abstract]
Stenmark Askmalm M, Stal O, Sullivan S, Ferraud L, Sun XF, Carstensen J, Nordenskjold B (1994) Cellular accumulation of p53 protein: an independent prognostic factor in stage II breast cancer. Eur J Cancer 30A:175-180
Sturzbecher HW, Brain R, Addison C, Rudge K, Remm M, Grimaldi M, Keenan E, Jenkins JR (1992) A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization. Oncogene 7:1513-1523[Medline]
Sun Y, Dong Z, Nakamura K, Colburn NH (1993) Dosage-dependent dominance over wild-type p53 of a mutant p53 isolated from nasopharyngeal carcinoma. FASEB J 7:944-950
Sun Y, Hegamyer G, Cheng YJ, Hildesheim A, Chen JY, Chen IH, Cao Y, Yao KT, Colburn NH (1992) An infrequent point mutation of the p53 gene in human nasopharyngeal carcinoma. Proc Natl Acad Sci USA 89:6516-6520[Abstract]
Waterhouse L, Muir C, Shanmugaratnam K, Powell J, Peachham D, Whelan S (1982) Cancer Incidence in Five Continents. Vol IV. Lyon, IARC Scientific Publications
Yu MC, Mo CC, Chong WX, Yeh FS, Henderson BE (1988) Preserved foods and nasopharyngeal carcinoma: a case-control study in Guangxi, China. Cancer Res 48:1954-1959[Abstract]
Zeng Y, Zhong JM, Mo YK, Miao XC (1983) Epstein-Barr virus early antigen induction in Raji cells by Chinese medicinal herbs. Intervirology 19:201-204[Medline]